Transdermal drug delivery systems

ABSTRACT

One aspect of the invention provides a transdermal delivery system including a drug formulated with a transport chaperone moiety that reversibly associates with the drug. The chaperone moiety is associated with the drug in the formulation so as to enhance transport of the drug across dermal tissue and releasing the drug after crossing said dermal tissue.

1. BACKGROUND OF THE INVENTION

Transdermal drug delivery offers a variety of advantages over oral andintravenous dosage. These include sustained release directly to thebloodstream over a long period of time, bypass of the gastrointestinaland hepatic elimination pathways, high patient compliance, and an easilyadministered dosage form that is portable and inexpensive.¹ Passive drugtransport across human skin is governed by Fick's Law of diffusion. Themass transport equation is given as:$J = {{\frac{1}{A}( \frac{\mathbb{d}M}{\mathbb{d}t} )} = {P\quad\Delta\quad C}}$where J is flux (μg cm⁻² hr⁻¹), A is cross sectional area of the skinmembrane (cm²), P is the apparent permeability coefficient (cm hr⁻¹), ΔCis the concentration gradient across the membrane, and (dM/dt) is themass transport rate. Research in the area of transdermal drug deliveryhas led to the commercial production of patches for nitroglycerin,²estrogen,³ testosterone,⁴ and nicotine.⁵ Although these represent majoradvances for transdermal delivery, these formulations rely primarily onthe natural diffusion of the drug from solution into the bloodstream.Most drugs show greatly reduced diffusivity through human skin, and thuswill not achieve therapeutic concentrations in the blood. Examples ofsuch drugs include large molecular weight drugs⁶ and ionic, hydrophilicdrugs.⁷¹Walters, K. A. In Penetration enhancers and their use in transdermaltherapeutic systems; Hadgraft, J.; Guy, R.; Eds.; Marcel Dekker: NewYork, 1989, 197-246²U.S. Pat. No. 5,670,164³U.S. Pat. No. 6,143,319⁴U.S. Pat. No. 5,422,119⁵U.S. Pat. No. 5,633,008⁶Mitragotri, Samir; Johnson, Mark E.; Blankschtein, Daniel; Langer,Robert. Biophysical Journal. 1999, 77, 1268-1283.⁷Gorukanti, Sudhir R.; Li, Lianli; Kim, Kwon H. Int. J. Pharm. 1999,192, 159-172

One method to counteract this drawback is to include chemical permeationenhancers. These components are soluble in the formulation and act toreduce the barrier properties of human skin. The list of potential skinpermeation enhancers is long, but can be broken down into three generalcategories: lipid disrupting agents (LDAs), solubility enhancers, andsurfactants. LDAs are typically fatty acid-like molecules proposed tofluidize lipids in the human skin membrane.^(8,9) Solubility enhancersact by increasing the maximum concentration of drug in the formulation,thus creating a larger concentration gradient for diffusion. Surfactantsare amphiphilic molecules capable of interacting with the polar andlipid groups in the skin.⁸Francoeur, Michael L.; Golden, Guia M.; Potts, Russell O. Pharm. Res.1990, 7, 621-627⁹U.S. Pat. No. 5,503,843

Another field of research drawn on by this invention pertains tomicroemulsion (ME) systems. Characteristics of such systems aresub-micron droplet size, thermodynamic stability, optical transparency,and solubility of both hydrophilic and hydrophobic components.¹⁰ MEsystems have been investigated as transdermal drug delivery vehicles,and have been found to exhibit improved solubility of hydrophobic drugsas well as sustained release profiles.¹⁰Lawrence, M. J., et. al. Int. Journal of Pharmaceutics. 1998, 111,63-72

Previous applications of chemical enhancers relevant to this inventioninclude formulations containing NMP,¹¹ oleic acid,¹² lidocaine,¹³ andmicroemulsion based systems.¹⁴¹¹U.S. Pat. No. 5,449,670¹²U.S. Pat. No. 6,106,856¹³U.S. Pat. No. 5,900,249¹⁴U.S. Pat. No. 5,833,647

SUMMARY OF THE INVENTION

One aspect of the invention provides a transdermal delivery systemincluding a drug formulated with a transport chaperone moiety thatreversibly associates with the drug. The chaperone moiety is associatedwith the drug in the formulation so as to enhance transport of the drugacross dermal tissue and releasing the drug after crossing said dermaltissue.

The chaperone moiety and drug can be associated, for example, by ionic,hydrophobic, hydrogen-bonding and/or electrostatic interactions. Incertain exemplary embodiments, the transport chaperone is n-methylpyrrolidone (NMP), octadecene, isopropyl myristate (IPM), oleyl alcohol,oleic acid or a derivative thereof.

In certain exemplary embodiments, the drug is a lidocaine, a prilocaine,an estradiol or a diltiazem. In certain exemplary embodiments, the drugis a free base, such as lidocaine HCl, lidocaine free base, prilocaineHCl, estradiol or diltiazem HCl.

Another aspect of the invention provides a microemulsion system fortransdermal delivery of a drug, which system solubilizes bothhydrophilic and hydrophobic components. For instance, the microemulsioncan be a cosolvent system including a lipophilic solvent and an organicsolvent. Exemplary cosolvents are NMP and IPM.

In certain preferred embodiments, the microemulsion system has anaqueous phase, a hydrophobic organic phase, and a surfactant phase. Forinstance, the microemulsion system has an aqueous phase of water andethanol, an organic phase of isopropyl mystate (IPM) and a surfactantphase of Tween 80. Another example of a microemulsion system has anaqueous phase of water and ethanol, an organic phase of IPM, and asurfactant phase is Tween 80 and Span 20.

In certain preferred embodiments, the microemulsion system is awater-in-oil system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Transport Chaperone Hypothesis

FIG. 2: Schematic of Drug Delivery from Multi-Phasic System

FIG. 3: NMP Chaperoning of Lidocaine Free Base from an Organic (IPM)Solvent

FIG. 4: NMP Chaperone of Lidocaine Free Base from an Aqueous (H₂O)Solvent

FIG. 5: ME System 1

FIG. 6: System 2

FIG. 7: System 3

FIG. 8: System 4

FIG. 9: Ethanol and NMP as Partitioning Agents

FIG. 10: IPM/NMP System for the Delivery of Lidocaine Free Base

FIG. 11: H₂O/NMP System for Lidocaine Free Base Delivery

FIG. 12: O/W ME Transport of Lidocaine Free Base Across Stripped HumanCadaver Skin

FIG. 13: O/W ME Transport of Lidocaine HCl Across Stripped Human CadaverSkin

FIG. 14: O/W ME Transport of Estradiol Across Stripped Human CadaverSkin

FIG. 15: O/W ME Transport of Diltiazem HCl Across Stripped Human CadaverSkin

FIG. 16: IPM/NMP Binary Vehicles Through Stripped Human Cadaver Skin

FIG. 17: Correlation of NMP and Lidocaine Steady State Flux AcrossStripped Human SC

FIG. 18: Correlation of NMP and Lidocaine Flux_(ss) in NMP Solvent withLDAs

FIG. 19: Lidocaine Free Base Flux Through Stripped Human Cadaver Skin inH₂O/NMP Cosolvent

FIG. 20: Phase Diagram of Water:Ethanol:IPM:Tween 80 Microemulsion

FIG. 21: Phase Diagram of Water:Ethanol:IPM:Tween 80:Span 20Microemulsion System

FIG. 22: Phase Diagram of Water:IPM:Tween 80:Ethanol MicroemulsionSystem

FIG. 23: Phase Diagram of Water:IPM:Tween 80:Ethanol MicroemulsionSystem

FIG. 24: Estradiol Transport Across Stripped Human Skin in MEFormulation

FIG. 25: Diltiazem HCl Transport Across Stripped Human Skin in MEFormulation

FIG. 26: Effect of NMP on Lidocaine Partitioning

DETAILED DESCRIPTION OF THE INVENTION

The novelty of our invention is the systematic incorporation ofpermeation enhancers to create robust drug delivery vehicles. There arethree key advances set forth in this invention. 1) A hypothesis drivenschematic by which transdermal drug delivery can be enhanced. 2) Theincorporation of proven permeability enhancers which meet thespecifications of the hypothesis, thus providing specific enhancementsystems based on the proposed schematic. 3) The enhanced delivery ofselect drugs utilizing the novel systems.

EXAMPLE 1 Chaperone-Mediated Transport for Transdermal Delivery

A. Mechanism to Enhance Transdermal Drug Delivery

The basis of our hypothesis for enhancement is the idea of a transportchaperone (FIG. 1). An ideal chaperone molecule should have thefollowing properties: high affinity to the drug, solubility in multiplevehicles, rapid permeation through the skin. The chaperone willreversibly bind to the drug molecule in the formulation. Because of theinherent permeation of the chaperone through the skin, it will be ableto “pull” the drug across the skin into the bloodstream. As the complexis diluted in the bloodstream, the interaction will reverse and the drugwill be released. In this model, a drug was chaperoned into and acrossthe skin. The same effect could also occur between the chaperone andanother permeation enhancer (such as LDA) to improve its effect.

Our hypothesis further extends to a biphasic formulation, namely an O/Wmicroemulsion. The advantage of having a biphasic system is the abilityto solubilize both hydrophilic and hydrophobic components. In thissystem, the hydrophobic drug must first leave the organic phase and intothe bulk aqueous phase (FIG. 2). This is accomplished through apartitioning agent which increases the concentration of drug in theouter, aqueous phase. Once in the aqueous phase, the chaperone describedabove can enhance transport through the skin. Notice that an aqueousphase chaperone is also capable of enhancing LDA activity andhydrophilic drugs from the O/W ME.

B. Proof of Principle

Our hypothesis was verified through in vitro drug flux studies acrossstripped human cadaver skin. For the permeation chaperone, n-methylpyrrolidone (NMP) was selected as the model molecule due to itsmiscibility with both organic and aqueous phases, its high drugsolubility, rapid flux through human skin (˜10 mg/cm²/hr), and hydrogenbonding capability (with free amine and hydroxyl groups). IRspectroscopy suggests that NMP forms hydrogen bonds with the amino groupof lidocaine free base. The correlation coefficient between lidocainefree base flux and NMP flux across human skin was large from both anorganic (isopropyl myristate, IPM) solvent (r²=0.97, FIG. 3) as well asfrom a H₂O solvent (FIG. 4, r²=0.93). This supports the claim that NMPis capable of acting as a transport chaperone for lidocaine free base.Further, the chaperoning of LDAs was tested in vitro. The enhancingeffects of the molecules octadecene, IPM, oleyl alcohol, and oleic acidwere determined. From an IPM bulk phase, none of these LDA-likemolecules had any permeation enhancement. However, when NMP was used asthe bulk solvent, there was a clear enhancement of lidocaine flux (Table1). NMP is necessary for the LDAs to have an enhancing effect onlidocaine flux. Furthermore, the molecules capable of hydrogen bondingwith NMP (oleyl alcohol and oleic acid have free hydroxyl groups) showsignificantly greater effect. This supports the claim that NMP aids LDAactivity through the chaperone hypothesis.

The multi-phase transport of our hypothesis was experimentallyevaluated. We first described novel ME systems with the followingcomponents (FIGS. 5-8). Organic System Aqueous Phase Phase SurfactantPhase 1 H₂O:Ethanol (1:1) IPM Tween 80 2 H₂O IPM Tween 80:Ethanol (1:1)3 H₂O IPM Tween 80:Ethanol (2:1) 4 H₂O:Ethanol (1:1) IPM Tween 80:Span20 (49:51)All ME systems were able to dissolve 10% w/w of NMP, oleyl alcohol, aswell as other enhancers, and a maximum load of ˜30% lidocaine free baseand ˜25% lidocaine HCl. Both NMP and ethanol were viable as partitionagents described above. These solvents were able to increase theconcentration of hydrophobic drugs in the aqueous phase and that ofhydrophilic drugs in the organic phase (FIG. 9). Further support of thishypothesis was observed in the finding that O/W microemulsions exhibitedgreater flux than W/O microemulsion, owing to the hypothesis that NMPworks primarily from the water phase. This is supported by the findingthat the [IPM]/[H₂O] partition coefficient of NMP is 0.02. Additionally,flux from just the water phase of the system (with the surfactants andorganic phase removed) is statistically equivalent to the O/W ME flux(Table 2). This indicates that the water phase is the dominant mode ofenhancement by NMP. Another interesting feature of the O/W system isthat the simultaneous delivery of both hydrophilic (diltiazem HCl) andhydrophobic (estradiol) drugs is not diminished from either drug alone(Table 2).C. Drug Transport Profiles

The systems described above were applied to the delivery of a number ofdrugs. Formulations utilizing the NMP transport chaperone principle werecreated in IPM and H₂O. Both the IPM/NMP system (FIG. 10) and theH₂O/NMP system (FIG. 11) showed improved drug delivery characteristicsfor the model hydrophobic drug lidocaine free base. The H₂O/NMP systemwas also found to be capable of providing enhancement for hydrophilicionic salt drugs (Table 3).

Both the W/O and O/W ME systems were evaluated for drug delivery. Thissystems was able to provide significant permeability enhancement for alldrugs tested (Table 4). Transport profiles for lidocaine free base (FIG.12), lidocaine HCl (FIG. 13), estradiol (FIG. 14), and diltiazem HCl(FIG. 15) indicate that steady state is reached in vitro at about 4hours.

D. Tables TABLE 1 NMP Synergy with LDAs IPM Solvent NMP SolventLidocaine Flux_(ss) Lidocaine Flux_(ss) NMP Flux_(ss) LDA (1% w/v)(μg/cm²/hr ± SD) (μg/cm²/hr ± SD) (mg/cm²/hr ± SD) None 1.95 ± 0.22 92 ±15 12.6 ± 0.5 Octadecene 1.98 ± 0.58 161 ± 52  15.3 ± 1.2 Isopropyl —232 ± 120 15.8 ± 3.6 Myristate Oleic Acid 1.68 ± 0.24 290 ± 103 17.6 ±2.0 Oleyl Alcohol 1.97 ± 0.48 402 ± 52  20.7 ± 1.0

TABLE 2 Estradiol and Diltiazem HCl Transport from ME Systems EstradiolDiltiazem HCl Formu- Flux_(ss) Permeability Flux_(ss) Permeabilitylation (μg/cm²/hr) (cm/hr · 10⁵) (μg/cm²/hr) (cm/hr · 10⁵) H₂O 0.015 ±0.006 460 ± 183 0.05 ± 0.01 0.015 ± 0.004 W/O ME 0.053 ± 0.029 1.1 ± 0.60.25 ± 0.13 1.2 ± 0.6 W/O ME 0.12 ± 0.06 2.4 ± 1.2 0.24 ± 0.08 1.2 ± 0.4Both Drugs O/W ME 0.27 ± 0.07 5.8 ± 1.5 1.6 ± 0.3 7.8 ± 1.3 O/W ME 0.23± 0.05 5.0 ± 1.2 1.6 ± 0.4 7.8 ± 1.9 Both Drugs Water 6.5 ± 1.7 6.1 ±3.7 Phase

TABLE 3 Flux Enhancement of Hydrophilic HCl Salt Drugs from 1:1 H2O/NMPCosolvent through Stripped Human Cadaver Skin Flux_(ss) from Flux_(ss)from H₂O H₂O/NMP Drug (2% w/v) (μg/cm²/hr ± SD) (μg/cm²/hr ± SD)Enhancement Lidocaine HCl 1.00 ± 0.22 4.35 ± 1.84 4.3 ± 2.1 PrilocaineHCl 2.44 ± 0.98 6.31 ± 0.14 2.6 ± 1.0

TABLE 4 Permeability Enhancement of ME Systems Permeability (cm/hr ·10⁵) Enhancement Estradiol IPM <0.1 W/O ME 1.1 ± 0.6 >11 O/W ME 5.8 ±1.5 >58 Diltiazem HCl H₂O 0.015 ± 0.004 W/O ME 1.2 ± 0.6 80 O/W ME 7.8 ±1.3 520 Lidocaine Free Base IPM 7.2 ± 1.1 W/O ME 40.1 ± 4.5  5.6 O/W ME123 ± 36  17 Lidocaine HCl H₂O 0.61 ± 0.38 W/O ME 3.5 ± 0.3 5.7 O/W ME18.1 ± 6.9  30E. References for Example 1

-   1. Walters, K. A. In Penetration enhancers and their use in    transdermal therapeutic systems; Hadgraft, J.; Guy, R.; Eds.; Marcel    Dekker: New York, 1989, 197-246-   2. U.S. Pat. No. 5,670,164-   3. U.S. Pat. No. 6,143,319-   4. U.S. Pat. No. 5,422,119-   5. U.S. Pat. No. 5,633,008-   6. Mitragotri, Samir; Johnson, Mark E.; Blankschtein, Daniel;    Langer, Robert. Biophysical Journal. 1999, 77, 1268-1283.-   7. Gorukanti, Sudhir R.; Li, Lianli; Kim, Kwon H. Int. J. Pharm.    1999, 192, 159-172-   8. Francoeur, Michael L.; Golden, Guia M.; Potts, Russell O. Pharm.    Res. 1990, 7, 621-627-   9. U.S. Pat. No. 5,503,843-   10. Lawrence, M. J., et. al. Int. Journal of Pharmaceutics. 1998,    111, 63-72-   11. U.S. Pat. No. 5,449,670-   12. U.S. Pat. No. 6,106,856-   13. U.S. Pat. No. 5,900,249-   14. U.S. Pat. No. 5,833,647

EXAMPLE 2 Evaluation of Chemical Enhancers in the Transdermal Deliveryof Lidocaine

Lidocaine free base is a local anesthetic routinely used in topicalapplications. This study aims at investigating the effect of variousclasses of chemical enhancers on in vitro drug transport across humanand pig skin. The lipid disrupting agents oleic acid, oleyl alcohol,butene diol, and decanoic acid show no significant flux enhancement. Thebinary system of isopropyl myristate/n-methyl pyrrolidone (IPM/NMP)exhibits a marked synergistic effect on drug transport. This effectpeaks at 25:75 v/v IPM:NMP reaching a steady state flux of 57.6±8.4 μgcm⁻² hr⁻¹ through human skin. This is 4-fold enhancement over a NMPsolution and over 25-fold increase over IPM (p<0.001). There is a tightcorrelation of lidocaine flux with NMP flux (r²=0.97) over the range ofNMP≦75%. IR spectroscopy analysis of lidocaine solutions indicates thatit forms hydrogen bonds in the presence of NMP solvent. This suggeststhat NMP may act as a “transport chaperone,” capable of enhancing theflux of drug molecules if delivered in the IPM/NMP system.

A. Introduction.

Lidocaine is a widely used local anesthetic for a variety of medicalprocedures including treatment of open skin sores and lesions, surgicalprocedures such as suturing of wounds, and venipuncture.¹⁵ Lidocaine isalso a first line anti-arrhythmic drug when administered to the heart inlarger doses.¹⁶ The most common method of lidocaine delivery is throughIV or hypodermic injection. When lidocaine is injected as an analgesicagent, the discomfort caused by the application is counterproductive tothe pain relieving effect of the drug. For purposes such as preparationfor pediatric venipuncture, a painless means to administer lidocaine tothe site of injection would be an important procedure. This makes localtransdermal delivery of lidocaine a likely avenue of research.Transdermal lidocaine products such as EMLA® cream (AstraZeneca) andLidoderm® (Endo Laboratories) are commercially available. However,further improvement in enhancement of transdermal lidocaine delivery isstill desired.¹⁵Smith, D W; Peterson, M R; DeBerard, S C. Postgraduate Medicine. 1999,106(2), 57-60, 64-66¹⁶Sleight, P. J. Cardiovasc. Pharmacol. 1990, 16, Suppl 5: S113-119

The primary barrier to transdermal drug delivery is the outermost layerof the skin, the stratum corneum (SC).¹⁷ The SC consists ofkeratinocytes embedded in a continuous lipid phase, forming a tortuousnetwork preventing the infiltration of exogenous agents into the body.¹⁸A variety of methods for increasing transdermal drug transport arecurrently studied. These include chemical enhancers,¹⁹ therapeutic andlow frequency ultrasound,²⁰ iontophoresis,²¹ and electroporation.²²While all these methods are capable of providing significant enhancementof drug delivery, a simple passive system free of additional machinerywould prove most effective for the local delivery of lidocaine. In thisstudy, the effects of a variety of chemical permeation enhancers areevaluated for the transdermal delivery of lidocaine free base.¹⁷Ranade, Vasant V. J. Clin. Pharmacol. 1991, 31, 401-418¹⁸Johnson, Mark E.; Blankschtein, Daniel; Langer, Robert. Journal ofPharmaceutical Sciences. 1997, 86, 1162-1172¹⁹Walters, K. A. In Penetration enhancers and their use in transdermaltherapeutic systems; Hadgraft, J.; Guy, R.; Eds.; Marcel Dekker: NewYork, 1989, 197-246²⁰Mitragotri, S.; Blankschtein, D.; Langer, R. Science. 1995, 269,850-853²¹Burnette, R. R. In Iontophoresis; Hadgraft, J.; Guy, R. H.; Eds.;Marcel Dekker: New York, 1989; 247-291²²Prausnitz, M. R.; Bose, V. G.; Langer, R.; Weaver, J. C. PNAS 1993,90, 10504-10508

Chemical enhancers with different proposed mechanisms of action weretested for their effects alone and in combination. Two main transportpathways have been proposed through human SC—the polar, aqueous pathwayand the lipid pathway.¹⁷ The majority of research on transdermal drugtransport to date has been focused on delivery through the continuouslipid region of the SC. Furthermore, transport of drug through theaqueous pathway has proven very difficult.²³ Since lidocaine free baseis a lipophilic molecule (log octanol-water partition coefficient=2.48),it is reasonable to explore lipid pathway enhancing chemicals to improvedrug flux. The more commonly studied chemical enhancers can be brokendown into 3 broad categories. The first is the class of lipid disruptingagents (LDAs), usually consisting of a long hydrocarbon chain with acis-unsaturated carbon-carbon double bond.^(24,25) These molecules havebeen shown to increase the fluidity of the SC lipids, thereby increasingdrug transport. In this study, oleic acid, oleyl alcohol, decanoic acid,and butene diol were investigated as lipid disrupting agents. A secondclass of permeation enhancers relies on improving drug solubility andpartitioning into the skin.²⁶ The lipophilic vehicle isopropyl myristate(IPM)²⁷ as well as the organic solvents ethanol²⁸ and N-methylpyrrolidone (NMP)²⁹ were studied. A final class of enhancers consists ofsurfactants. These molecules have affinity to both hydrophilic andhydrophobic groups, which might facilitate in traversing the complexregions of the SC. An anionic surfactant lauryl sulfate (SDS) and anonionic surfactant polysorbate 80³⁰ (Tween 80) was tested for theireffect on lidocaine delivery.¹⁷Ranade, Vasant V. J. Clin. Pharmacol. 1991, 31, 401-418²³Peck, Kendall D.; Ghanem, Abdel-Halem; Higuchi, William I. J. Pharm.Sci. 1995, 84, 975-982²⁴Francoeur, Michael L.; Golden, Guia M.; Potts, Russell O. Pharm. Res.1990, 7, 621-627²⁵Kim, Dae-Duk; Chien, Yie W. J. Pharm. Sci. 1996, 85, 214-219²⁶Guy, Richard H.; Hadgraft, Jonathan. J. Controlled Release. 1987, 5,43-51²⁷Gorukanti, Sudhir R.; Li, Lianli; Kim, Kwon H. Int. J. Pharm. 1999,192, 159-172²⁸Liu, Puchun; Kurihara-Bergstrom, Tamie; Good, William R. Pharm. Res.1991, 8, 938-944²⁹Yoneto, Kunio; Li, S. Kevin; Ghanem, Abdel-Halim; Crommelin, Daan J.A.; Higuchi, William I. J. Pharm. Sci. 1995, 84, 853-860³⁰Sarpotdar, Pramod P.; Zatz, Joel L. J. Pharm. Sci. 1986, 75, 176-181

It has been reported in the literature that combinations of variousenhancers result in a synergistic increase in drug flux that is fargreater than either chemical by itself.^(31,32) Various combinations ofenhancer combinations were tested to identify useful trends in lidocainefree base delivery. Because of the different solubility of lidocainefree base in each of the delivery vehicles, saturated solutions wereutilized in each sample to maintain a constant thermodynamic activity ofthe drug.³¹Johnson, Mark E.; Mitragotri, Samir; Patel, Ashish; Blankschtein,Daniel; Langer, Robert. J. Pharm. Sci. 1996, 85, 670-679³²Sasaki, Hitoshi; Kojima, Masaki; Nakamura, Junzo; Shibasaki, Juichiro.J. Pharm. Pharmacol. 1990, 42, 196-199

Lidocaine free base is a commonly studied drug for transdermaldelivery^(17,33) as its hydrophobicity and molecular size (MW 234.3)characterize it as a typical transdermal drug candidate. By studying theeffects of a wide range of chemical enhancers across both full thicknesspig skin and stripped human cadaver skin, some general trends oftransdermal permeation enhancement can be hypothesized.¹⁷Ranade, Vasant V. J. Clin. Pharmacol. 1991, 31, 401-418³³Johnson, Mark E., Blankschtein, Daniel; Langer, Robert. J. Pharm. Sci.1995, 84, 1144-1146

B. Materials

Drug: Lidocaine free base was purchased from Sigma (St. Louis, Mo.).Chemicals: NMP was a generous gift from ISP Technologies, Inc. (Wayne,N.J.). USP grade oleic acid was purchased from Mednique. Polysorbate 80NF (Tween 80) was purchased from Advance Scientific & Chem. (Ft.Lauderdale, Fla.). Isopropyl myristate (IPM), oleyl alcohol (99%),anhydrous ethyl alcohol, SDS, cis-2-butene-1,4-diol, decanoic (capric)acid, and phosphate buffered saline tablets (PBS) were purchased fromSigma (St. Louis, Mo.). HPLC grade solvents were used as received. Skin:Human cadaver skin from the chest, back, and abdominal regions wasobtained from the National Disease Research Institute (Philadelphia,Pa.). The skin was stored at −80° C. until use.

C. Methods

(i) Preparation of Lidocaine Solutions. Sample solutions were preparedin 20 ml glass vials and saturated with drug. In binary systems thesample contained 50% (w/w) of each liquid. All vehicles studied formedmiscible, single phase liquids.

(ii) Determination of Saturation Concentration. All samples were mixedwith a magnetic stir-bar in the presence of lidocaine free base crystalsfor at least 24 hours at room temperature. The saturated solutions werethen syringe filtered through a 0.2 μm filter to remove undissolveddrug. Concentrations of the filtered solutions were determined by HPLCafter dilution to a suitable range.

(iii) Preparation of Skin Samples. Human cadaver skin was thawed at roomtemperature. The epidermis-SC was separated from the full thicknesstissue after immersion in 60° C. water for 2 minutes. Heat stripped skinwas immediately mounted on diffusion cells. Full thickness pig skin wasprepared by removing the dermal tissue from a freshly sacrificed pig.Pig skin samples were subsequently frozen and stored at −20° C. or −80°C.

(iv) Lidocaine Transport Experiments. The skin was mounted onto aside-by-side glass diffusion cell with an inner diameter of 5 mm. Thetwo halves of the cell were clamped shut and both reservoirs were filledwith 2 ml of phosphate buffered saline (PBS, 0.01 M phosphate, 0.137 MNaCl, pH 7.4). The integrity of the skin was verified by measuring theelectrical conductance across the skin barrier at 1 kHz and 10 Hz at143.0 mV (HP 33120A Waveform Generator). Skin samples measuring 4-14 μAat 1 kHz were used for the diffusion studies. Prior to introducing thedonor solution, the skin sample was thoroughly rinsed with PBS to removesurface contaminants. At t=0, the receiver compartment was filled with2.0 ml of PBS, while 2.0 ml of sample was added to the donorcompartment. Both compartments were continuously stirred to maintaineven concentrations. At regular time intervals, 1.0 ml of the receivercompartment was transferred to a glass HPLC vial. The remaining solutionin the receiver compartment was thoroughly aspirated and discarded.Fresh PBS (2.0 ml) was dispensed into the receiver compartment tomaintain sink conditions. At 21 hours, the experiment was terminated.After both compartments were refilled with PBS, the conductance acrossthe skin membrane was again checked to ensure that the skin was notdamaged during the experiment. All flux experiments were conducted intriplicate at room temperature. The observed variability of theindividual drug transport values was consistent with the previouslyestablished 40% intersubject variability of human skin.³⁴³⁴Williams, A. C.; Cornwell, P. A.; Barry, B. W. Int. J. Pharm. 1992,86, 69-77

(v) IPM/NMP Binary Vehicle Transport. The two miscible liquids weremixed in the specified v/v ratios, with 2% w/v lidocaine free baseadded. Flux cells were set up as described above. At t=4, 21, 23, 25hours, the transport of drug across the skin was measured by HPLC.Steady state conditions were taken as the average of the final 2 timepoints.

(vi) Quantification of Lidocaine. Lidocaine was assayed by high pressureliquid chromatography (Shimadzu model HPLC, SCL-10A Controller, LC-10ADpumps, SPD-M10A Diode Array Detector, SIL-10AP Injector, Class VPv.5.032 Integration Software) on a reverse phase column (WatersμBondapak™ C₁₈ 3.9×150 mm) using ddH₂O (5% acetic acid, pH4.2)/acetonitrile (35:65 v/v) as the mobile phase, under isocraticconditions (1.6 mL/min) by detection at 237 nm. The retention time oflidocaine under these conditions was between 3.4 and 4.3 minutes.Standard solutions were used to generate calibration curves. NMP wasquantified on a Waters Symmetry® C₁₈ 5 μm, 3.9×150 mm column(WAT046980). The mobile phase consisted of ddH₂O:methanol (95:5) at aflow rate of 1.2 ml/min. Chromatograms were integrated at a peak of 205nm, with retention time at 3.8-4.8 min.

(vii) Calculations. The total mass of drug transported across the skinwas determined by HPLC. The flux equation gives:$F = {{\frac{1}{A}( \frac{\mathbb{d}M}{\mathbb{d}t} )} = {P\quad\Delta\quad C}}$

where F is flux (μg cm⁻² hr⁻¹), A is cross sectional area of the skinmembrane (cm²), P is the apparent permeability coefficient (cm hr⁻¹),and ΔC is the concentration gradient. In this experiment, ΔC is taken asthe saturation concentration (given infinite dose and sink conditions),and dM/dt is averaged as the total mass transport over the time courseof the experiment. Statistical analyses were performed by the Student'st-test.

D. Results

(i) Lidocaine Free Base Solubility. The maximum concentration of drug inan application vehicle is an important element determining transdermalflux. According to Fick's Law of diffusion, flux across a membranescales linearly with concentration. The maximum level of drug transportacross the skin should occur when the donor solution is saturated withdrug. The solubility of a drug in various solutions also givesindications of molecular interactions such as hydropobicity, hydrogenbonding capability, and pH dependence.

Due to the lipophilicity of lidocaine free base, its solubility inaqueous media was limited. The saturation concentration of lidocainefree base in water was over 60 times less than its solubility in ahydrocarbon oil such as isopropyl myristate (IPM). Although the additionof the anionic surfactant SDS (CMC=0.2%) at 10 mg/ml improved thesolubility of the hydrophobic drug in water, the saturation pointremained too low to provide appreciable flux. The solubility oflidocaine free base in the hydrophobic enhancers was in the 300-400mg/ml range (Table 5). The two solvents that significantly improved thesaturation concentration of lidocaine free base were ethanol (618 mg/ml)and NMP (733 mg/ml).

(ii) Permeability of Lidocaine Free Base. The in vitro permeability oflidocaine free base across stripped human skin and full thickness pigskin gives an indication of the enhancing effect of each chemical beyondtheir ability to improve drug saturation concentration. The permeabilityof lidocaine free base in saturated neat enhancer solutions is given inTable 5. Although the apparent permeability of lidocaine from the twoaqueous solutions (H₂O and 1% SDS) appear significant compared to theother samples, they should be discounted as viable delivery vehicles dueto their minimal lidocaine saturation point. The physical flux oflidocaine free base across human skin in vitro from water was 10.8±1.95μg cm⁻² hr⁻¹ as compared to 20.4±3.02 μg cm⁻² hr⁻¹ from an IPM solution.This significant difference (p<0.001) makes water a poor candidate as atransdermal vehicle. A similar trend was seen in full thickness pigskin.

(iii) LDAs. The chemical enhancers with lipid-disrupting ability (oleylalcohol, oleic acid, butene-diol) did not show significant improvementof lidocaine permeability or flux. In fact, the flux and permeability oflidocaine free base in the presence of these enhancers was eitherstatistically equivalent or below that of IPM solution.

(iv) Solubility/Partition Enhancers. Permeability experiments acrossboth human and pig skin indicated that the two chemical enhancers withhighest drug solubility were poor permeation enhancers. The permeabilityof lidocaine free base in saturated solutions of ethanol and NMP did notsurpass that of IPM. By themselves, these two solvents appeared to beable to increase drug flux only by improving drug solubility. These skinpenetration enhancers were unable to markedly increase lidocaine freebase permeability as neat enhancer solutions.

(v) Surfactants. The two surfactant enhancers studied (SDS andpolysorbate 80) did not significantly increase drug flux. Among the twoaqueous donors (H₂O and 1% SDS), there was no statistical differencebetween the permeability of lidocaine through human or pig skin. SDS wascapable of increasing the solubility of lidocaine free base, but it hadno noticeable enhancing effect on skin permeability. Polysorbate 80tended to increase the viscosity of the solution and had a severelynegative effect on drug transport (data not shown).

(vi) Permeability in Cosolvent Systems. To evaluate possible synergisticinteraction between the studied enhancers, 1:1 ratios of selectedchemicals were mixed to form cosolvent systems (Table 6). It has beenpreviously reported that combining transdermal chemical enhancers cangreatly improve drug transport through human skin.³⁵ For practicalpurposes, IPM was selected as the bulk oil phase to be mixed with otherenhancers. It is a molecule consisting of a long hydrocarbon chain,satisfying the hypothesis that lidocaine free base is transported bestthrough a hydrophobic vehicle. IPM is also inexpensive, easy to workwith, and exhibited the best in vitro permeability of the studiedenhancers. Cosolvents of IPM were made with oleyl alcohol, oleic acid,decanoic acid, and NMP. Decanoic acid is chemically similar to oleicacid, and may act in the skin as a lipid disrupting agent. In thesecosolvent systems the LDAs had no enhancing effect on lidocaine freebase permeability. The trend appears similar to that of the neatsolvents, suggesting that mixing these LDAs with IPM did not result inpronounced enhancement. The only IPM/cosolvent system that had asignificant effect on permeability across stripped human skin was theIPM/NMP system (p<0.005). Because of the higher drug solubility of thisNMP containing system, the total transdermal flux across human skin(165±27 μg cm⁻² hr⁻¹) was roughly 8-fold greater than that of just anIPM vehicle, and over 6-fold better than a saturated NMP system(p<0.001). A similar trend was seen in full thickness pig skin (Table6).³⁵Priborsky, Jan; Takayama, Kozo; Nagai, Tsuneji; Waitzova, Danuse;Elis, Jiri. Drug Design and Delivery. 1987, 2, 91-97

(vii) IPM/NMP Cosolvent Flux. From these experiments, an IPM/NMPcosolvent system appeared to exhibit the best flux. To furtherinvestigate this effect, the two enhancers were mixed in varying ratiosin the presence of 2% lidocaine free base (Table 7). Between 10% and 75%NMP concentration, the increase in lidocaine flux scaled linearly withNMP concentration. Lidocaine flux was equivalent at 75% and 90% NMP(p>0.5), and increasing to 100% NMP reduced flux to 26% of its previousvalue (FIG. 16). When the flux of NMP through the skin of the samevehicles was tested, it produced similar results (FIG. 17). In the rangeof linear flux increase (10-75% NMP), there was a corresponding linearincrease in NMP flux (r²=0.97). When NMP concentration was increased to90% and 100%, the total NMP flux from the vehicle decreased.

(viii) IR Analysis of Lidocaine Systems. To gain a better understandingof why IPM/NMP is a good transdermal enhancer for lidocaine, IR spectraof drug solution in various vehicles was obtained. From the saturationstudy, it was established that NMP is the best lidocaine free basesolvent of the chemicals studied. However, the permeability from theIPM/NMP cosolvent system suggests that there was an effect other thanimproving donor concentration. In IR spectra of lidocaine free base inNMP, the amide group band was shifted lower, suggesting hydrogen bondingby NMP. The potential of NMP to form hydrogen bonds with the amide groupof lidocaine may facilitate the transport of drug through SC.

E. Discussion

This evaluation of chemical permeation enhancers on lidocaine free basetransport across human skin expands the current knowledge of theeffectiveness of transdermal enhancers. There currently exists a largecollection of chemicals believed to enhance transdermal drug delivery,yet their benefits have been difficult to apply broadly to multipledrugs. In this study, lidocaine free base was selected as a model drugto aid in the understanding of the relative effectiveness of some of themore widely used chemical enhancers.

Due to the continuous lipid regions in the stratum corneum, it iscurrently believed that passive transdermal diffusion occurspredominantly through the lipid phase of the skin.^(3,4) For thisreason, hydrophobic drugs generally have better transport through skinwhile water soluble ionic drugs have very limited permeability.³⁶ Thisrelation holds true for lidocaine free base. Because of its limitedsolubility in water, the amount of drug transported from this phase isless than from an oil phase vehicle. Lidocaine also exists as ahydrophilic hydrochloride salt (lidocaine HCl). As expected, both theflux and the permeability of lidocaine HCl through human skin is anorder of magnitude less than for the free base (Data not shown). Basedon these observations, we decided to investigate lipophilic enhancers.³U.S. Pat. No. 6,143,319⁴U.S. Pat. No. 5,422,119³⁶Lee, Cheon Koo; Uchida, Takahiro; Kitagawa, Kazuhisa; Yagi, Akira;Kim, Nak-Seo; Goto, Shigeru. J. Pharm. Sci. 1994, 83, 562-565

A common class of such enhancers is the LDAs. These hydrophobicmolecules are believed to fluidize the stratum corneum lipids and reduceits barrier properties. In the case of lidocaine free base, none ofthese agents improved the permeability of drug across the skin.Saturated solutions of oleyl alcohol, oleic acid, butene-diol, anddecanoic acid yielded very poor lidocaine free base transport. (1% oleylalcohol in IPM also had no statistical improvement.) This result mightbe explained by the hypothesis that transport of relatively smallmolecules such as lidocaine free base are not severely hindered by lipidbilayers.^(17,37,38) Altering the bilayer properties in this case willnot result in dramatic flux increases. In order to enhance lidocainepermeability, an enhancer must target a step of the transport processthat is rate determining.¹⁷Ranade, Vasant V. J. Clin. Pharmacol. 1991, 31, 401-418³⁷Mitragotri, Samir; Johnson, Mark E.; Blankschtein, Daniel; Langer,Robert. Biophysical Journal. 1999, 77, 1268-1283.³⁸Mitragotri, Samir. Pharm. Res. 2001, 18(7), 1018-23

NMP and its derivatives are widely used chemical enhancers which haveproduced significant results in the transport of various drugs.^(18,39)More recently, they have been used in conjunction with more lipophilicmolecules to enhance partitioning of more hydrophilic drugs into theskin.¹⁵ Although NMP by itself is an exceptional solvent for drugs, ourexperiments show that it does not greatly improve the permeability oflidocaine free base. However, combining NMP with IPM results is insubstantial flux improvement. In 2% lidocaine systems, the maximum fluxoccurs between 75% and 90% NMP, with a linear relationship below 75%NMP. This relationship may be useful by allowing the control of drugflux by adjusting NMP concentration in the vehicle.¹⁸Johnson, Mark E.; Blankschtein, Daniel; Langer, Robert. Journal ofPharmaceutical Sciences. 1997, 86, 1162-1172³⁹Phillips, Christine A.; Michniak, Bozena B. J. Pharm. Sci. 1995, 84,1427-1433¹⁵Smith, D W; Peterson, M R; DeBerard, S C. Postgraduate Medicine. 1999,106(2), 57-60, 64-66

The synergy observed in saturated IPM/NMP systems in vitro could beexplained by an osmotic pressure gradient into the donor compartmentthat retards the flux of drug. NMP is freely miscible in water, and hasa high affinity for it. An osmotic gradient is created between the donorcompartment (>700 mg/ml lidocaine free base) and the receivercompartment (PBS). Since water diffuses through human skin fairlyreadily, the osmotic pressure will drive water from the receiver intothe salt rich donor compartment. We observed that lidocaine crystalsprecipitate from the donor compartment as it becomes infused with water.This reverse gradient could explain the reduced lidocaine transport froma saturated NMP solution. A rationale for the synergy of the IPM/NMPcosolvent system is the hydrophobic nature of IPM. Since IPM is notmiscible with water, its presence in the donor compartment might deterthe flux of water across the skin. In the absence of this osmoticpressure effect (2% lidocaine load), NMP shows improved transportproperties. At this concentration, the permeability is over 25-foldbetter than saturated NMP.

At low drug concentration (2% lidocaine), a different IPM/NMPinteraction likely occurs (FIG. 16). Below a threshold of ˜10% NMP,there is little effect on drug flux enhancement. Above thisconcentration, NMP permeability reaches a high level, and remainsconstant in the 10% to 75% NMP range. Increasing NMP beyond this levelquickly diminishes permeability. The reason for this is unclear, but maystem from molecular interactions between NMP, IPM, and lidocaine. Apossible hypothesis is that when NMP is close to 100% of the solution,it forms solvent-solvent bonds which make leaving the donor compartmentunfavorable. When the non-polar IPM is introduced, it disrupts theseinteractions and pushes the NMP equilibrium into the donor. The exactmechanism is an avenue of further research.

From our experiments, we conclude that NMP is the preferred transdermalenhancer. Its method of action is most likely through improving thepartitioning of lidocaine free base through the SC barrier. This processmay be facilitated by hydrogen bonding between NMP and the drug, assuggested by IR spectroscopy. Experimentally, there was strongcorrelation between NMP flux and lidocaine flux. This raises thepossibility that NMP may act as a “molecular chaperone” to enhance drugdelivery. NMP displays very high permeability through human SC (1.8•10⁻²cm hr⁻¹ in the NMP/IPM systems), which may serve as a driving force forlidocaine free base flux. This same property should also apply to otherdrugs which hydrogen bond with NMP.

F. Tables TABLE 5 Effect of Chemical Enhancers on Lidocaine TransportStripped Human Cadaver Skin Full Thickness Pig Skin Time Averaged TimeAveraged Saturation Time Averaged Flux Time Averaged Flux ConcentrationPermeability (μg · cm⁻² · hr⁻¹) ± Permeability (μg · cm⁻² · hr⁻¹) ±Sample (mg/ml) (cm hr⁻¹ · 10⁵) ± SD SD (cm hr⁻¹ · 10⁵) ± SD SD H₂O 4.5238 ± 43  10.8 ± 1.95 6.93 ± 3.46 0.496 ± 0.248 1% SDS 8.3 246 ± 16821.3 ± 14.6 9.29 ± 3.60 0.804 ± 0.312 in H₂O IPM 246 7.17 ± 1.06 20.4 ±3.02 0.515 ± 0.117 0.847 ± 0.192 NMP 733 2.92 ± 0.42 26.8 ± 3.86 0.120 ±0.007 0.706 ± 0.043 Oleyl 361 3.68 ± 1.59 14.5 ± 6.28 — — Alcohol OleicAcid 428 0.61 ± 0.28 4.04 ± 1.83 — — Butene 386 1.18 ± 0.47 4.56 ± 1.80— — Diol Ethanol 618 5.14 ± 3.55 31.7 ± 21.9 0.0649 ± 0.0190 0.182 ±0.053

TABLE 6 Effect of 1:1 Cosolvent Systems on Lidocaine Transport StrippedHuman Cadaver Skin Full Thickness Pig Skin Time Averaged Time AveragedSaturation Time Averaged Flux Time Averaged Flux ConcentrationPermeability (μg · cm⁻² · hr⁻¹) ± Permeability (μg · cm⁻² · hr⁻¹) ±Sample (mg/ml) (cm hr⁻¹ · 10⁵) ± SD SD (cm hr⁻¹ · 10⁵) ± SD SD IPM 2467.17 ± 1.06 20.4 ± 3.02 0.515 ± 0.117 0.847 ± 0.192 IPM/NMP 365 20.0 ±6.92 53.4 ± 18.5 1.37 ± 0.69 3.64 ± 1.83 9:1 IPM/NMP 641 18.9 ± 3.10 165 ± 27.1 0.965 ± 0.521 2.78 ± 1.50 1:1 NMP 733 2.92 ± 0.42 26.8 ±3.86 0.120 ± 0.007 0.706 ± 0.043 IPM/Oleyl 345 3.62 ± 1.12 17.7 ± 5.48 —— Alcohol IPM/Oleic 355 4.93 ± 0.57 32.5 ± 3.73 — — Acid IPM/Decanoic309 2.47 ± 1.50 7.64 ± 4.65 — — Acid

TABLE 7 Lidocaine Transport from IPM/NMP Cosolvent Systems ThroughStripped Human Cadaver Skin Lidocaine NMP 24 Hour 24 Hour % NMP NMPFlux_(ss) Transport Flux_(ss) Transport (v/v) (mg/ml) (μg cm⁻² hr⁻¹) ±SD (μg) ± SD (μg cm⁻² hr⁻¹) ± SD (μg) ± SD 0 0  1.98 ± 0.57 31.4 ± 6.4 0 0 10 105  4.69 ± 0.84 99.7 ± 25.7 1.46 ± 0.9 22.8 ± 3.7 25 259 16.4 ±0.5 443 ± 73   5.40 ± 0.49 83.6 ± 5.7 50 518 32.5 ± 4.0 731 ± 179  9.01± 0.84  112 ± 5.3 60 620 40.7 ± 1.2 729 ± 72  11.2 ± 1.2 134 ± 13 75 77856.7 ± 4.9 1040 ± 50  14.0 ± 0.9 161 ± 6  90 935 57.6 ± 8.4 907 ± 2 11.7 ± 0.1 128 ± 1  100 1040 15.4 ± 0.6 333 ± 25  10.7 ± 0.2  115 ± 0.2G. Conclusion

NMP is capable of enhancing transdermal delivery by chaperoninglidocaine across human skin. The maximum lidocaine flux occurs from asolution of 25:75 IPM/NMP. There is strong correlation between NMP fluxand lidocaine flux across skin samples. It is hypothesized that NMPparticipates in drug transport via hydrogen bonding. The high lidocaineflux obtained from the IPM/NMP cosolvent is a promising indication ofthe utility of this vehicle for transdermal drug delivery.

H. References for Example 2

-   15. Smith, D W; Peterson, M R; DeBerard, S C. Postgraduate Medicine.    1999, 106(2), 57-60, 64-66-   16. Sleight, P. J. Cardiovasc. Pharmacol. 1990, 16, Suppl 5:    S113-119-   17. Ranade, Vasant V. J. Clin. Pharmacol. 1991, 31, 401-418-   18. Johnson, Mark E.; Blankschtein, Daniel; Langer, Robert. Journal    of Pharmaceutical Sciences. 1997, 86, 1162-1172-   19. Walters, K. A. In Penetration enhancers and their use in    transdermal therapeutic systems; Hadgraft, J.; Guy, R.; Eds.; Marcel    Dekker: New York, 1989, 197-246-   20. Mitragotri, S.; Blankschtein, D.; Langer, R. Science. 1995, 269,    850-853-   21. Burnette, R. R. In Iontophoresis; Hadgraft, J.; Guy, R. H.;    Eds.; Marcel Dekker: New York, 1989; 247-291-   22. Prausnitz, M. R.; Bose, V. G.; Langer, R.; Weaver, J. C. PNAS    1993, 90, 10504-10508-   23. Peck, Kendall D.; Ghanem, Abdel-Halem; Higuchi, William I. J.    Pharm. Sci. 1995, 84, 975-982-   24. Francoeur, Michael L.; Golden, Guia M.; Potts, Russell O. Pharm.    Res. 1990, 7, 621-627-   25. Kim, Dae-Duk; Chien, Yie W. J. Pharm. Sci. 1996, 85, 214-219-   26. Guy, Richard H.; Hadgraft, Jonathan. J. Controlled Release.    1987, 5, 43-51-   27. Gorukanti, Sudhir R.; Li, Lianli; Kim, Kwon H. Int. J. Pharm.    1999, 192, 159-172-   28. Liu, Puchun; Kurihara-Bergstrom, Tamie; Good, William R. Pharm.    Res. 1991, 8, 938-944-   29. Yoneto, Kunio; Li, S. Kevin; Ghanem, Abdel-Halim; Crommelin,    Daan J. A.; Higuchi, William I. J. Pharm. Sci. 1995, 84, 853-860-   30. Sarpotdar, Pramod P.; Zatz, Joel L. J. Pharm. Sci. 1986, 75,    176-181-   31. Johnson, Mark E.; Mitragotri, Samir; Patel, Ashish;    Blankschtein, Daniel; Langer, Robert. J. Pharm. Sci. 1996, 85,    670-679-   32. Sasaki, Hitoshi; Kojima, Masaki; Nakamura, Junzo; Shibasaki,    Juichiro. J. Pharm. Pharmacol. 1990, 42, 196-199-   33. Johnson, Mark E., Blankschtein, Daniel; Langer, Robert. J.    Pharm. Sci. 1995, 84, 1144-1146-   34. Williams, A. C.; Cornwell, P. A.; Barry, B. W. Int. J. Pharm.    1992, 86, 69-77-   35. Priborsky, Jan; Takayama, Kozo; Nagai, Tsuneji; Waitzova,    Danuse; Elis, Jiri. Drug Design and Delivery. 1987, 2, 91-97-   36. Lee, Cheon Koo; Uchida, Takahiro; Kitagawa, Kazuhisa; Yagi,    Akira; Kim, Nak-Seo; Goto, Shigeru. J. Pharm. Sci. 1994, 83, 562-565-   37. Mitragotri, Samir; Johnson, Mark E.; Blankschtein, Daniel;    Langer, Robert. Biophysical Journal. 1999, 77, 1268-1283.-   38. Mitragotri, Samir. Pharm. Res. 2001, 18(7), 1018-23-   39. Phillips, Christine A.; Michniak, Bozena B. J. Pharm. Sci. 1995,    84, 1427-1433

EXAMPLE 3 Chaperone-Mediated Transport for Transdermal Delivery

The interaction between chemical enhancers in a transdermal formulationis crucial to its function. In this study, n-methyl pyrrolidone (NMP)was studied as a water phase enhancer for lidocaine free base transport.It has been proposed that NMP acts as a flux chaperone via hydrogenbonding with solutes. This paper supports this hypothesis by findingthat lipid disrupting agents (LDAs) capable of hydrogen bonding with NMPprovide better lidocaine free base flux than analogous non-hydrogenbonding molecules. It was also found that NMP is capable of thechaperone effect above 50% v/v in H₂O for lidocaine free base. Additionof the LDA oleic acid improved flux up to 6-fold in the presence of NMP(35.1 μg/cm²/hr). The H₂O/NMP (50% v/v) system increased the flux of thehydrophilic ionic salt drugs lidocaine HCl and prilocaine HCl 4.3 and2.6 fold, respectively. These findings support the NMP chaperonehypothesis and suggest that NMP is capable of enhancing hydrophilic,ionic drugs from an aqueous solution.

A. Introduction

Transdermal drug delivery is a promising route for the administration oftherapeutic agents to the bloodstream painlessly and in a controlledmanner.^(40,41) Current methods which have been developed to improvetransdermal transport include chemical enhancers,⁴² therapeutic and lowfrequency ultrasound,⁴³ iontophoresis,⁴⁴ and electroporation.⁴⁵ It iscrucial to investigate passive chemical enhancer systems because oncefavorable chemical interactions are found, they can be applied to theother means of transdermal enhancement.⁴⁶ Theoretical frameworks havebeen proposed to explain the effects of molecular size,⁴⁷diffusion,^(48,49) and partitioning^(50,51) across bilayer membranes.However, one of the more complex parameters affecting transdermal drugdelivery is the interaction of the constituents in the enhancerformulation.⁵² Although combining chemical enhancers often results ingreatly improved drug flux,^(53,54) the mechanism of this effect isoften difficult to determine. This paper will focus on the chemicalenhancer n-methyl pyrrolidone (NMP) as a flux chaperone as well as itsrole in an aqueous solvent.⁴⁰Asbill, C S; El-Kattan, A F; Michniak, B. Crit. Rev. Ther. DrugCarrier Syst. 2000, 17(6), 621-58⁴¹Ranade, Vasant V. J. Clin. Pharmacol. 1991, 31, 401-418⁴²Walters, K. A. In Penetration enhancers and their use in transdermaltherapeutic systems; Hadgraft, J.; Guy, R.; Eds.; Marcel Dekker: NewYork, 1989, 197-246⁴³Mitragotri, S.; Blankschtein, D.; Langer, R. Science. 1995, 269,850-853⁴⁴Burnette, R. R. In Iontophoresis; Hadgraft, J.; Guy, R. H.; Eds.;Marcel Dekker: New York, 1989; 247-291⁴⁵Prausnitz, M. R.; Bose, V. G.; Langer, R.; Weaver, J. C. PNAS 1993,90, 10504-10508⁴⁶Mitragotri, Samir. Pharm. Res. 2000, 17(11), 1354-58⁴⁷Mitragotri, Samir; Johnson, Mark E.; Blankschtein, Daniel; Langer,Robert. Biophysical Journal. 1999, 77, 1268-1283.⁴⁸Johnson, Mark E.; Blankschtein, Daniel; Langer, Robert. Journal ofPharmaceutical Sciences. 1997, 86, 1162-1172⁴⁹Johnson, Mark E.; Berk, David A.; Blankschtein, Daniel; Golan, DavidE.; Jain, Rakesh K.; Langer, Robert. Biophysical Journal. 1996, 71,2656-2668.⁵⁰Phillips, Christine A.; Michniak, Bozena B. J. Pharm. Sci. 1995, 84,1427-1433⁵¹Lee, Cheon Koo; Uchida, Takahiro; Kitagawa, Kazuhisa; Yagi, Akira;Kim, Nak-Seo; Goto, Shigeru. J. Pharm. Sci. 1994, 83, 562-565⁵²Pugh, W. J.; Hadgraft, J.; Roberts, M. S. Int. J. Pharm. 1996, 138,149-65⁵³Johnson, Mark E.; Mitragotri, Samir; Patel, Ashish; Blankschtein,Daniel; Langer, Robert. J. Pharm. Sci. 1996, 85, 670-679⁵⁴Sasaki, Hitoshi; Kojima, Masaki; Nakamura, Junzo; Shibasaki, Juichiro.J. Pharm. Pharmacol. 1990, 42, 196-199

Although there is no broad theoretical model predicting solute-solventinteractions in transdermal flux, it has been shown in previousexperiments that strong relationships can exist.^(55,56) Results acrossmodel membranes indicate that solute flux is proportional to solventuptake. Furthermore, this parameter can be predicted based onsolubility, MW, and hydrogen bonding interactions. Our previous workwith NMP suggests that it is capable of enhancing lidocaine free basetransport across human skin. It has been hypothesized that this ismediated through hydrogen bonding between NMP and the amide group oflidocaine free base. When in the presence of the highly lipophilicsolvent isopropyl myristate (IPM), NMP exhibits significant enhancementproperties for lidocaine free base transport. It was also found that thedegree of drug flux correlated closely with the amount of NMP fluxacross the skin. This finding supported the hypothesis that NMP can actas a chaperone for transdermal transport across human skin. An importantquestion addressed by the current study is whether NMP in a water phasesolvent is capable of the same enhancement.⁵⁵Liu, Puchun; Kurihara-Bergstrom, Tamie; Good, William R. Pharm. Res.1991, 8, 938-944⁵⁶Cross, Sheree; Pugh, W. John; Hadgraft, Jonathan; Roberts, Michael S.Pharm. Res. 2001, 18(7) 999-1005

An enhancer capable of improving drug flux from the water phase wouldgreatly expand the current repertoire of transdermally deliverabledrugs. The primary barrier to drug transport across human skin is thestratum corneum.⁵⁷ Due to the nature of this lipid membrane, aqueousphase transport has proven difficult.^(58,59,60) NMP was investigated asa possible water phase flux enhancer due to its miscibility with waterand earlier reports of improved transdermal delivery.^(61,62,63)Additionally, based on the hypothesis of hydrogen bonding between NMPand formulation solutes as a means of enhancement, the effect of lipiddisrupting agents (such as oleic acid)^(64,65) should also be aided byNMP. The flux enhancement of analogous 18 carbon molecules withdifferent end groups (and thus differing hydrogen bonding capacity) wasused to test this hypothesis. Lidocaine free base was selected as themodel drug as its interaction with NMP has been previously studied. Itcan also be derived as a water soluble, ionic salt (lidocaine HCl). NMPsystems were further investigated to determine whether they are capableof providing enhancement of the hydrophilic ionic drugs lidocaine HCland prilocaine HCl.⁵⁷Barry, B. W.; Bennett, S. L. J. Pharm. Pharmacol. 1987, 39, 535-46⁵⁸Gorukanti, Sudhir R.; Li, Lianli; Kim, Kwon H. Int. J. Pharm. 1999,192, 159-172⁵⁹Roy, Samir D.; Roos, Eric; Sharma, Kuldeepak. J. Pharm. Sci. 1994, 83,126-130⁶⁰Peck, Kendall D.; Ghanem, Abdel-Halem; Higuchi, William I. J. Pharm.Sci. 1995, 84, 975-982⁶¹Sasaki, Hitoshi; Kojima, Masaki; Nakamura, Junzo; Shibasaki, Juichiro.J. Pharm. Pharmacol. 1990, 42, 196-199⁶²Phillips, Christine A.; Michniak, Bozena B. J. Pharm. Sci. 1995, 84,1427-1433⁶³Yoneto, Kunio; Li, S. Kevin; Ghanem, Abdel-Halim; Crommelin, Dann J.A.; Higuchi, William I. J. Pharm. Sci. 1995, 84(7), 853-61⁶⁴Francoeur, Michael L.; Golden, Guia M.; Potts, Russell O. Pharm. Res.1990, 7, 621-627⁶⁵Kim, Dae-Duk; Chien, Yie W. J. Pharm. Sci. 1996, 85, 214-219

B. Materials

Drugs: Lidocaine free base, lidocaine HCl, and prilocaine HCl werepurchased from Sigma (St. Louis, Mo.). Chemicals: NMP was a generousgift from ISP Technologies, Inc. (Wayne, N.J.). USP grade oleic acid waspurchased from Mednique. Isopropyl myristate (IPM), 9-octadecene, oleylalcohol (99%), anhydrous ethyl alcohol, and phosphate buffered salinetablets (PBS) were purchased from Sigma (St. Louis, Mo.). HPLC gradesolvents were used as received. Skin: Human cadaver skin from the chest,back, and abdominal regions was obtained from the National DiseaseResearch Institute (Philadelphia, Pa.). The skin was stored at −80° C.until use.

C. Methods

(i) Preparation of Drug Solutions. Sample solutions were prepared in 20ml glass vials and loaded with drug. All vehicles studied formedmiscible, single phase liquids.

(ii) Preparation of Skin Samples. Human cadaver skin was thawed at roomtemperature. The epidermis-SC was separated from the full thicknesstissue after immersion in 60° C. water for 2 minutes. Heat stripped skinwas immediately mounted on diffusion cells.

(iii) Transport Experiments. The skin was mounted onto a side-by-sideglass diffusion cell with an inner diameter of 5 mm. The two halves ofthe cell were clamped shut and both reservoirs were filled with 2 ml ofphosphate buffered saline (PBS, 0.01 M phosphate, 0.137 M NaCl, pH 7.4).The integrity of the skin was verified by measuring the electricalconductance across the skin barrier at 1 kHz and 10 Hz at 143.0 mV (HP33120A Waveform Generator). Skin samples measuring 4-10 μA at 1 kHz wereused for the diffusion studies. Prior to introducing the donor solution,the skin sample was thoroughly rinsed with PBS to remove surfacecontaminants. At t=0, the receiver compartment was filled with 2.0 ml ofPBS, while 2.0 ml of sample was added to the donor compartment. Bothcompartments were continuously stirred to maintain even concentrations.At regular time intervals, 1.0 ml of the receiver compartment wastransferred to a glass HPLC vial. The remaining solution in the receivercompartment was thoroughly aspirated and discarded. Fresh PBS (2.0 ml)was dispensed into the receiver compartment to maintain sink conditions.At 24 hours, the experiment was terminated. After both compartments wererefilled with PBS, the conductance across the skin membrane was againchecked to ensure that the skin was not damaged during the experiment.All flux experiments were conducted in triplicate at room temperature.The observed variability of the individual drug transport values wasconsistent with the previously established 40% intersubject variabilityof human skin.⁶⁶⁶⁶Williams, A. C.; Cornwell, P. A.; Barry, B. W. Int. J. Pharm. 1992,86, 69-77

(iv) LDA Chaperoning. IPM solutions of 2% (w/v) lidocaine free base and1% (w/v) LDA were mixed in 20 ml glass vials. The LDA's includedoctadecene, oleyl alcohol, and oleic acid. Similar solutions with NMP asthe solvent were also obtained. The flux of lidocaine free base and ofNMP through human cadaver skin was determined as detailed above.

(v) H₂O/NMP Binary Vehicle Transport. The two miscible liquids weremixed in the specified v/v ratios, with 2% w/v lidocaine free baseadded. Flux cells were set up as described above. At t=20, 22, 24 hours,the transport of drug across the skin was measured by HPLC. Steady stateconditions were taken as the average of the final 2 time points. After24 hours, the drug solution was removed and the skin rinsed with PBS.Two ml of the equivalent H₂O/NMP mixture with 1% oleic acid (v/v) wasthen added to the donor compartment of the flux cell. The effect of thissolution on drug transport was measured by HPLC after an additional 6,7, and 8 hours.

(vi) Hydrophilic Drug Transport The water soluble drugs lidocaine HCland prilocaine HCl were dissolved in 2% (w/v) doses in 1:1 H₂O:NMP(v/v). Flux through stripped human skin was compared with flux of thesame drugs through distilled water. After 24 hours, oleic acid (1%) wasintroduced to the donor solutions of the lidocaine HCl samples. All drugconcentrations were analyzed by HPLC.

(vii) NMP Patitioning. Distilled water (2 ml), IPM (2 ml), and NMP (40μl) were thoroughly vortexed in a glass tube. After equilibrating for 1hour, the sample was centrifuged at 14,000 rpm for 6 minutes andseparated into 2 phases. Samples of each phase were taken to determineNMP concentration by HPLC.

(viii) Quantification of Transport. Lidocaine was assayed by highpressure liquid chromatography (Shimadzu model HPLC, SCL-10A Controller,LC-10AD pumps, SPD-M10A Diode Array Detector, SIL-10AP Injector, ClassVP v.5.032 Integration Software) on a reverse phase column (WatersμBondapak™ C₁₈ 3.9×150 mm) using ddH₂O (5% acetic acid, pH4.2)/acetonitrile (35:65 v/v) as the mobile phase, under isocraticconditions (1.6 mL/min) by detection at 237 nm. The retention time oflidocaine under these conditions was between 3.4 and 4.3 minutes.Standard solutions were used to generate calibration curves. The sameHPLC method was utilized for prilocaine HCl, with the exception that itwas measured at 254 nm. NMP was quantified on a Waters Symmetry® C₁₈ 5μm, 3.9×150 mm column (WAT046980). The mobile phase consisted ofddH₂O:methanol (95:5) at a flow rate of 1.2 ml/min. Chromatograms wereintegrated at a peak of 205 nm, with retention time at 3.8-4.8 min.

(ix) Calculations. The total mass of drug transported across the skinwas determined by HPLC. The flux equation gives:$J = {{\frac{1}{A}( \frac{\mathbb{d}M}{\mathbb{d}t} )} = {P\quad\Delta\quad C}}$where J is flux (μg cm⁻² hr⁻¹), A is cross sectional area of the skinmembrane (cm²), P is the apparent permeability coefficient (cm hr⁻¹),and ΔC is the concentration gradient. In this experiment, ΔC is taken asthe donor concentration (assuming infinite dose and sink conditions),and dM/dt is averaged as the total mass transport over the time courseof the final two time points. Statistical analyses were performed by theStudent's t-test.D. Results

(i) LDA Chaperoning. Flux of lidocaine free base through human cadaverskin from IPM solutions is given in Table 8. There is no statisticalsignificance (p>0.40) among any of the IPM samples. When NMP is used asthe bulk solvent, there is a trend of increasing lidocaine free baseflux from no LDA, octadecene, IPM, oleic acid, oleyl alcohol. All fourlipid compounds improve drug flux over neat NMP (p<0.05). The bestenhancer, oleyl alcohol is statistically better than IPM and octadecene(p<0.05). The flux of NMP from these solutions closely matches that oflidocaine transport, with a R² value of 0.93 (FIG. 18).

(ii) H₂O/NMP Binary Cosolvent. Lidocaine free base (2% w/v) transportwas investigated in varying combinations of H₂O/NMP (Table 9). Becauselidocaine free base is sparsely soluble in water, the 80%, 90%, and 100%H₂O samples were saturated below 2% drug. Both the flux and permeabilityof drug at varying NMP concentration results in a V-shaped curve. NMPdoes not begin transporting across the skin unless it is above ˜50% ofthe donor solution. Plotting the NMP and lidocaine fluxes for % NMP≧50%results in a strong correlation (R² value of 0.98). When 1% oleic acidis used, the flux of the hydrophobic drug increases. The samples withgreater NMP flux (in the absence of oleic acid) showed improvedenhancement when oleic acid is introduced. Specifically, at 40% NMP(poor NMP flux), oleic acid had no effect (p>0.5) while at 80% NMP theflux enhancement was over 6-fold (35.1 μg/cm²/hr).

(iii) Hydrochloride Salt Transport. The flux of the hydrochloride saltdrugs lidocaine HCl and prilocaine HCl were investigated in H₂O/NMP (50%v/v). NMP appears to be capable of improving the flux of the two drugsroughly 2-4 fold (Table 10). The addition of 1% oleic acid to H₂O/NMP indoes not affect lidocaine HCl flux (p>0.5). When an IPM/NMP (50% v/v)system was utilized, the lidocaine HCl flux was even greater (15.7±7.9μg/cm²/hr). This was accompanied by a significantly larger NMP fluxthrough the skin.

(iv) IPM/H₂O Partitioning of NMP. The relative concentration of NMP wasdetermined from an equilibrium mixture of H₂O and IPM. NMP was found topartition 98% in the water phase (IPM/H₂O=0.02±0.001).

E. Discussion

The role of NMP as an aqueous phase transdermal chemical enhancer wasstudied in this experiment. We tested one hypothesis that NMP acts as achaperone molecule, facilitating solute transport into and across humanskin via hydrogen bonding. Interactions among formulation components isan important field of study in transdermal drug delivery. Although someresearch has been done regarding hydrogen bonding between solutes andartificial membranes,⁶⁷ flux enhancement as a result of hydrogen bondingbetween two co-transported species is not well understood.⁶⁷Du Plessis, Jeanetta; Pugh, W. John; Judefeind, Anja; Hadgraft,Jonathan. Eur. J. Pharm. Sci. 2001, 13, 135-141

It is proposed that NMP is capable of improving the efficacy ofenhancing agents such as LDAs. Based on molecular structures, oleic acidand oleyl alcohol should have greater hydrogen bonding capacity with NMPthan octadecene and IPM. The free hydroxyl groups of these two moleculesare capable of hydrogen bonding with the NMP oxygen. Table 8 indicatesthat all 4 lipid disrupting-like agents have statistically equivalenteffects on lidocaine free base transport from an IPM solvent. From theseresults, it is clear that none of these chemicals have an enhancingeffect on lidocaine flux from the hydrophobic solvent. However, if thesame agents are used in conjunction with an NMP solvent, there is adefinite enhancing effect. The addition of each of the LDA-likemolecules (1% v/v) improve drug flux over neat NMP solution (p<0.05). Itis also apparent that both lidocaine free base and NMP flux from thesesystems follows the trend predicted by hydrogen bonding, with flux fromoleic acid and oleyl alcohol systems being greater than IPM oroctadecene. This suggests that from an IPM solution, the hydrophobicLDAs are incapable of affecting the skin membrane. One possibleexplanation is that the LDAs have such high affinity for the donorsolvent that they do not partition into the skin. Once they are in theenvironment of a polar solvent (NMP), the LDAs are capable ofpartitioning into the hydrophobic stratum corneum. When present in theskin, the lipid disrupting agents are thought to reduce the barrierproperties of the stratum corneum, and improve drug permeability bycreating disorder in the lipids.²⁵ The subsequent enhancement in bothNMP flux and lidocaine free base flux can be explained by this effect ofthe LDAs.²⁵Kim, Dae-Duk; Chien, Yie W. J. Pharm. Sci. 1996, 85, 214-219

Both NMP and the LDAs enhance the transport of each of the othercomponents in the formulation, resulting in significantly improved drugflux. Although the exact mechanism is not clear, the results from thisexperiment can be explained in the following manner. The LDAs are in anenvironment favorable to their partitioning into the skin. As NMP has ahigh inherent flux through skin (12.6±0.5 mg/cm²/hr), it is able tofacilitate the transport of the LDA into the skin via the hypothesis ofhydrogen bonding. This, in turn, reduces the barrier properties of theskin, and improves NMP and drug flux. As NMP flux increases, it willcause an increase in drug flux due to its proposed chaperoning ability.This is supported by the high correlation (r²=0.93) between NMP andlidocaine free base flux in the formulations. Even with the largedifferences in LDAs and drug flux across the samples, there exists atight relationship between the flux of NMP and drug, supporting theclaim that there is indeed an interaction between the two molecules.

When NMP was used in conjunction with a water solvent, the resultsdiffered from that obtained with IPM/NMP. Two important characteristicsof the synergy curve are markedly different: the magnitude and shape.The steady state flux of lidocaine free base from an H₂O/NMP system issignificantly lower than an IPM/NMP system. The finding that NMP flux islower from water than from IPM is not surprising. Because NMP partitionsalmost exclusively into the water phase from an IPM mixture([IPM]/[H₂O]=0.02), it is much more likely to leave a donor solution ofIPM into a receiver compartment of PBS than to partition from water toPBS. In the water phase, NMP flux is driven only by a concentrationgradient. However, when IPM is the bulk solvent, the partitioning of NMPserves as an additional driving force.

From a water phase system (Table 9), it appears that NMP has essentiallyzero flux out of the formulation until it reaches a concentration ofabout 50% (v/v). This might be explained by the fact that NMP has veryhigh affinity for water. In small quantities, NMP may be completelysolvated by water and thermodynamically unable to partition into thehydrophobic skin membrane. Even at 50% (v/v) NMP, there is still a 6:1molar excess of water molecules in the system. Only when NMP exists atsuitable concentrations is it free to transport across the skin. From50-100% NMP, there is a steady increase in NMP flux across the skin(FIG. 19). In this range, it is observed that lidocaine free base fluxalso increases proportionally to NMP flux (r²=0.98). This supports thehypothesis that NMP acts as a drug chaperone. At and below 50% NMP, whenits flux is negligible, there is no enhancement of lidocaine flux. Infact, the data seems to support that from 0-50% (v/v) NMP, lidocaineflux is retarded by increasing NMP. Lidocaine free base has a highpermeability from water (13.3±2.3•10⁻⁴ cm/hr), and the addition of NMPreduces this value by 98% (0.24±0.05•10⁻⁴ cm/hr) at a concentration of50% NMP. The interaction of NMP with water limits the permeability oflidocaine free base across human skin. A possible explanation of thisresult is that in the absence of NMP flux across the skin, thehydrophobic lidocaine free base molecules have increased affinity forthe donor phase (with NMP as a solvent) and are less likely to partitioninto the skin. Further evidence of the NMP chaperone hypothesis is givenby the observation that oleic acid is ineffective as an enhancing agentunless there is significant NMP flux through the skin. This isconsistent with the finding in Table 8 where the absence of NMP fluxrenders the LDA ineffective.

Although the H₂O/NMP system is not as beneficial as IPM/NMP in improvingthe flux of the model drug lidocaine free base, its transport doessupport the hypothesis that NMP is capable of acting as a chaperone inthe water phase. This claim is further supported by the transport of thehydrophilic drugs lidocaine HCl and prilocaine HCl from the H₂O/NMPsystem. Both of these drugs are highly water soluble ionic compounds,making transdermal transport difficult.⁶⁸ From the data (Table 10), itappears that H₂O/NMP is capable of providing some flux enhancement forthese drugs. Interestingly, when oleic acid (1% v/v) was added to thelidocaine HCl sample, the flux was unaffected (p>0.5). A possiblehypothesis suggested by this observation is that the transport oflidocaine HCl goes through a pathway which is not limited by lipidbilayers. If this is the case, then NMP may be able to act as anenhancer in the aqueous transport pathway as well as the more commonlystudied lipid route. Although the extent of this enhancement may beimproved in numerous was, the finding that NMP can be effective inimproving the delivery of hydrophilic, ionic drugs opens up a wide areaof investigation.⁶⁸Peck, Kendall D.; Ghanem, Abdel-Halem; Higuchi, William I. J. Pharm.Sci. 1995, 84, 975-982

F. Tables TABLE 8 Lidocaine and NMP Flux with LDA IPM Solvent NMPSolvent Lidocaine Flux_(ss) Lidocaine Flux_(ss) NMP Flux_(ss) LDA (1%w/v) (μg/cm²/hr ± SD) (μg/cm²/hr ± SD) (mg/cm²/hr ± SD) None 1.95 ± 0.2292 ± 15 12.6 ± 0.5 Octadecene 1.98 ± 0.58 161 ± 52  15.3 ± 1.2 Isopropyl— 232 ± 120 15.8 ± 3.6 Myristate Oleic Acid 1.68 ± 0.24 290 ± 103 17.6 ±2.0 Oleyl Alcohol 1.97 ± 0.48 402 ± 52  20.7 ± 1.0N = 3

TABLE 9 Transport of Lidocaine and NMP from H₂O/NMP Binary SystemsLidocaine Free Base NMP Flux_(ss) Permeability Flux_(ss) (μg/cm²/ (cm/hr· (μg/cm²/hr ± Permeability (cm/ % NMP hr ± SD) 10⁴ ± SD) SD) hr · 10⁴ ±SD) 0 6.02 ± 1.04 13.3 ± 2.3  — — 10 5.36 ± 0.83 5.43 ± 0.84 0.007 ±0.007 1.08 ± 1.08 20 4.27 ± 1.20 2.86 ± 0.80 0.008 ± 0.003 0.64 ± 0.2340 2.15 ± 1.13 1.08 ± 0.56 0.026 ± 0.021 1.01 ± 0.83 50 0.47 ± 0.10 0.24± 0.05 0.010 ± 0.002 0.31 ± 0.05 60 1.95 ± 2.07 0.97 ± 1.03 0.106 ±0.058 2.73 ± 1.48 80 5.75 ± 0.95 2.87 ± 0.48 3.26 ± 0.74 62.8 ± 14.2 9011.4 ± 1.0  5.71 ± 0.51 9.18 ± 0.53 157 ± 9  100 15.4 ± 0.6  7.69 ± 0.2912.6 ± 0.5  63.1 ± 2.5 N = 3

TABLE 10 Flux Enhancement of Hydrophilic HCl Salt Drugs from 1:1 H2O/NMPCosolvent through Stripped Human Cadaver Skin Flux_(ss) from Flux_(ss)from H₂O H₂O/NMP Drug (2% w/v) (μg/cm²/hr ± SD) (μg/cm²/hr ± SD)Enhancement Lidocaine HCl 1.00 ± 0.22 4.35 ± 1.84 4.3 ± 2.1 PrilocaineHCl 2.44 ± 0.98 6.31 ± 0.14 2.6 ± 1.0N = 3G. Conclusion

This paper supports the claim that NMP acts as a transdermal enhancerthrough its hydrogen bonding capability with other formulation solutes.NMP was found to act in synergy with LDAs capable of hydrogen bonding(such as oleic acid and oleyl alcohol) to improve lidocaine free baseflux. These same LDAs had no effect from IPM and H₂O solutions,suggesting that the presence of NMP is central to their enhancementability. More specifically, it is crucial that NMP flux from the systembe appreciable for LDA effectiveness. The enhancement of oleic acid onlidocaine free base flux was negligible from H₂O/NMP systems where therewas no NMP flux (Table 9) as well as from IPM/NMP systems (˜10% NMP)where NMP flux was minimal (data not shown).

NMP also appears to be capable of providing drug delivery enhancementfrom the aqueous phase. In this paper, H₂O/NMP systems resulted inimproved LDA (oleic acid) effect, hydrophobic drug flux (lidocaine freebase), and hydrophilic ionic salt drug flux (lidocaine HCl andprilocaine HCl). All of these results are consistent with the hypothesisthat NMP behaves as a transdermal chaperone, acting through its hydrogenbonding capacity and high flux through the skin.

H. References for Example 3

-   40. Asbill, C S; El-Kattan, A F; Michniak, B. Crit. Rev. Ther. Drug    Carrier Syst. 2000, 17(6), 621-58-   41. Ranade, Vasant V. J. Clin. Pharmacol. 1991, 31, 401-418-   42. Walters, K. A. In Penetration enhancers and their use in    transdermal therapeutic systems; Hadgraft, J.; Guy, R.; Eds.; Marcel    Dekker: New York, 1989, 197-246-   43. Mitragotri, S.; Blankschtein, D.; Langer, R. Science. 1995, 269,    850-853-   44. Burnette, R. R. In Iontophoresis; Hadgraft, J.; Guy, R. H.;    Eds.; Marcel Dekker: New York, 1989; 247-291-   45. Prausnitz, M. R.; Bose, V. G.; Langer, R.; Weaver, J. C. PNAS    1993, 90, 10504-10508-   46. Mitragotri, Samir. Pharm. Res. 2000, 17(11), 1354-58-   47. Mitragotri, Samir; Johnson, Mark E.; Blankschtein, Daniel;    Langer, Robert. Biophysical Journal. 1999, 77, 1268-1283.-   48. Johnson, Mark E.; Blankschtein, Daniel; Langer, Robert. Journal    of Pharmaceutical Sciences. 1997, 86, 1162-1172-   49. Johnson, Mark E.; Berk, David A.; Blankschtein, Daniel; Golan,    David E.; Jain, Rakesh K.; Langer, Robert. Biophysical Journal.    1996, 71, 2656-2668.-   50. Phillips, Christine A.; Michniak, Bozena B. J. Pharm. Sci. 1995,    84, 1427-1433-   51. Lee, Cheon Koo; Uchida, Takahiro; Kitagawa, Kazuhisa; Yagi,    Akira; Kim, Nak-Seo; Goto, Shigeru. J. Pharm. Sci. 1994, 83, 562-565-   52. Pugh, W. J.; Hadgraft, J.; Roberts, M. S. Int. J. Pharm. 1996,    138, 149-65-   53. Johnson, Mark E.; Mitragotri, Samir; Patel, Ashish;    Blankschtein, Daniel; Langer, Robert. J. Pharm. Sci. 1996, 85,    670-679-   54. Sasaki, Hitoshi; Kojima, Masaki; Nakamura, Junzo; Shibasaki,    Juichiro. J. Pharm. Pharmacol. 1990, 42, 196-199-   55. Liu, Puchun; Kurihara-Bergstrom, Tamie; Good, William R. Pharm.    Res. 1991, 8, 938-944-   56. Cross, Sheree; Pugh, W. John; Hadgraft, Jonathan; Roberts,    Michael S. Pharm. Res. 2001, 18(7) 999-1005-   57. Barry, B. W.; Bennett, S. L. J. Pharm. Pharmacol. 1987, 39,    535-46-   58. Gorukanti, Sudhir R.; Li, Lianli; Kim, Kwon H. Int. J. Pharm.    1999, 192, 159-172-   59. Roy, Samir D.; Roos, Eric; Sharma, Kuldeepak. J. Pharm. Sci.    1994, 83, 126-130-   60. Peck, Kendall D.; Ghanem, Abdel-Halem; Higuchi, William I. J.    Pharm. Sci. 1995, 84, 975-982-   61. Sasaki, Hitoshi; Kojima, Masaki; Nakamura, Junzo; Shibasaki,    Juichiro. J. Pharm. Pharmacol. 1990, 42, 196-199-   62. Phillips, Christine A.; Michniak, Bozena B. J. Pharm. Sci. 1995,    84, 1427-1433-   63. Yoneto, Kunio; Li, S. Kevin; Ghanem, Abdel-Halim; Crommelin,    Dann J. A.; Higuchi, William I. J. Pharm. Sci. 1995, 84(7), 853-61-   64. Francoeur, Michael L.; Golden, Guia M.; Potts, Russell O. Pharm.    Res. 1990, 7, 621-627-   65. Kim, Dae-Duk; Chien, Yie W. J. Pharm. Sci. 1996, 85, 214-219-   66. Williams, A. C.; Cornwell, P. A.; Barry, B. W. Int. J. Pharm.    1992, 86, 69-77-   67. Du Plessis, Jeanetta; Pugh, W. John; Judefeind, Anja; Hadgraft,    Jonathan. Eur. J. Pharm. Sci. 2001, 13, 135-141-   68. Peck, Kendall D.; Ghanem, Abdel-Halem; Higuchi, William I. J.    Pharm. Sci. 1995, 84, 975-982

EXAMPLE 4 Microemulsion Enhancer Formulation for SimultaneousTransdermal Delivery of Hydrophilic and Hydrophobic Drugs

Microemulsion (ME) systems allow for the microscopic incorporation ofaqueous and organic phase liquids. In this study, the phase diagrams offour novel ME systems were characterized. Water and IPM composed theaqueous and organic phases respectively, while Tween 80 served as ananionic surfactant. Transdermal enhancers such as n-methyl pyrrolidone(NMP) and oleyl alcohol were incorporated into all systems withoutdisruption of the stable emulsion. A comparison of a W/O ME with an O/WME of the same system for lidocaine delivery indicated that an O/W MEprovides significantly greater flux (p<0.025). This finding, that thewater phase is a crucial component is consistent with in vitro fluxexperiments using hydrophobic drugs (lidocaine free base, estradiol) aswell as hydrophilic drugs (lidocaine HCl, diltiazem HCl). Furthermore,the simultaneous delivery of both a hydrophilic drug and a hydrophobicdrug from the ME system is indistinguishable from either drug alone.Enhancement of drug permeability from the O/W ME system was 17-fold forlidocaine free base, 30-fold for lidocaine HCl, 58-fold for estradiol,and 520-fold for diltiazem HCl.

A. Introduction

Microemulsions (ME) are thermodynamically stable emulsions with dropletsizes in the sub-micron range. They typically consist of an aqueousphase, an organic phase, and a surfactant/cosurfactant component. Thedesign and properties of microemulsion systems is a field that has beenstudied extensively with applications in many pharmaceutical areas.⁶⁹There are two basic types of ME systems: water-in-oil (W/O) andoil-in-water (O/W). In each case, it is believed that the minority phaseis encapsulated by the continuous bulk phase. Surfactants are necessaryto reduce the hydrophobic interactions between the phases and maintain asingle phase. Typical properties of ME include optical transparency,thermodynamic stability, and solubility of both hydrophobic andhydrophilic components.⁶⁹Lawrence, M. J., et. al. Int. Journal of Pharmaceutics. 1998, 111,63-72

Microemulsions have been proposed to offer enhanced drug deliveryproperties for transdermal transport.^(70,71) Flux enhancement fromthese formulations was found to be primarily due to an increase in drugconcentration. In these studies, it was concluded that drug transportoccurs only from the continuous (outer) phase. By this account,hydrophobic drugs transport faster from W/O emulsions, while O/W systemsprovide slower, controlled release of drug that is dependant on thepartitioning of drug into the outer phase. This pathway of drug releasefrom ME systems is supported by work with a hydrophilic molecule(glucose) where it was found to parallel the diffusion of water from thebulk phase.⁷² The stability and encapsulation properties of emulsionsmake the transdermal delivery of protein drugs an idealapplication.^(73,74,75,76)⁷⁰Trotta, M.; Gasco, M. R.; Morel, S. Journal of Controlled Release.1989, 10, 237-243⁷¹Trotta, M.; Pattarino, F.; Gasco, M. R. Pharmaceutica Acta Helvetiae.1996, 71, 135-140⁷²Osborne, D. W.; Ward, A. J. I.; O'Neill, K. J. J. Pharm. Pharmacol.1991, 43, 451-454⁷³Ho, Hsiu-O; Hsiao, Chih-Chuan; Sheu, Ming-Thau. J. Pharm. Sci. 1996,85(2) 138-143⁷⁴Guo, Jianxin; Ping, Qineng; Sun, Guoqin; Jiao, Chunhong. Int. J.Pharm. 2000, 194, 201-207⁷⁵Zhang, Qiang; Yie, Guoqing; Li, Yie; Yang, Qingsong; Nagai, T. Int. J.Pharm. 2000, 200, 153-159⁷⁶Baca-Estrada, Maria E.; Foldvari, Marianna; Ewen, Catherine; Badea,Ildiko; Babiuk, Lorne A. Vaccine. 2000, 18, 1847-1854

In this study, multiple features were incorporated into a MEformulation. Nonionic surfactants were selected to minimize skinirritation and charge disruption of the system. The main surfactantstudied, Tween 80 (Polysorbate 80) has previously been utilized intransdermal formulations.^(77,78) A key feature of the ME systemsstudied is incorporation of the transdermal chemical enhancers oleylalcohol and n-methyl pyrrolidone (NMP), which to our knowledge has neverbeen explored. Oleyl alcohol is a cis-unsaturated C₁₈ fatty acid whichis believed to reduce the barrier properties of the skin by disruptinglipid bilayers within the stratum corneum.^(79,80) NMP has been utilizedas a transdermal enhancer for multiple drugs and formulationcompositions, but never in conjunction with a ME.^(81,82,83) We selectedNMP based on our earlier studies showing that it is capable ofsignificantly enhancing drug transport from both the organic⁸⁴ andaqueous⁸⁵ phase. These findings supported our hypothesis that thehydrogen bonding capability of NMP with certain drugs, along with thehigh flux of NMP through human skin (˜10 mg/cm²/hr) allows NMP to act asa molecular chaperone. We propose that this enhancing ability shouldoccur in ME systems as well.⁷⁷Walters, K. A; Dugard, P. H., Florence, A. T. J. Pharm. Pharmacol.1981, 33, 207-213⁷⁸Sarpotdar, Pramod P.; Zatz, Joel L. J. Pharm. Sci. 1986, 75, 176-181⁷⁹Francoeur, Michael L.; Golden, Guia M.; Potts, Russell O. Pharm. Res.1990, 7, 621-627⁸⁰Kim, Dae-Duk; Chien, Yie W. J. Pharm. Sci. 1996, 85, 214-219⁸¹Sasaki, Hitoshi; Kojima, Masaki; Nakamura, Junzo; Shibasaki, Juichiro.J. Pharm. Pharmacol. 1990, 42, 196-199⁸²Phillips, Christine A.; Michniak, Bozena B. J. Pharm. Sci. 1995, 84,1427-1433⁸³Yoneto, Kunio; Li, S. Kevin; Ghanem, Abdel-Halim; Crommelin, Dann J.A.; Higuchi, William I. J. Pharm. Sci. 1995, 84(7), 853-61⁸⁴Lee, Philip J.; Ahmad, Naina; Mitragotri, Samir; Langer, Robert,Shastri, V. Prasad. “Evaluation of Chemical Enhancers in the TransdermalDelivery of Lidocaine.” Submitted Pharm. Res.⁸⁵Lee, Philip J.; Mitragotri, Samir; Langer, Robert, Shastri, V. Prasad.“Chaperoning of Lipid Disrupting Agents and Aqueous Phase TransdermalEnhancement by n-Methyl Pyrrolidone.” Submitted Pharm. Res.

In this study we have evaluated the transdermal transport of severalhydrophobic and hydrophilic drug moieties from novel ME systems thatincorporate chemical enhancers. Drug molecules investigated includelidocaine free base^(86,87) and HCl salt, estradiol^(88,89) anddiltiazem HCl, a drug which has not been previously studied in theliterature due to its large molecular weight (415 Da) and ionic,hydrophilic nature.⁸⁶Johnson, Mark E.; Mitragotri, Samir; Patel, Ashish; Blankschtein,Daniel; Langer, Robert. J. Pharm. Sci. 1996, 85, 670-679⁸⁷Johnson, Mark E., Blankschtein, Daniel; Langer, Robert. J. Pharm. Sci.1995, 84, 1144-1146⁸⁸Chien, Yie W.; Chien, Te-yen; Bagdon, Robert E.; Huang, Yih C.;Bierman, Robert H. Pharm. Res. 1989, 12, 1000-1010⁸⁹Powers, Marilou S.; Schenkel, Lotte; Darley, Paul E.; Good, WilliamR.; Balestra, Joanne C.; Place, Virgil A. Am. J. Obstet. Gynecol. 1985,152, 1099-1106

B. Materials

Drugs: Lidocaine free base, Lidocaine HCl, Estradiol, and Diltiazem HClwere purchased from Sigma (St. Louis, Mo.). Chemicals: NMP was agenerous gift from ISP Technologies, Inc. (Wayne, N.J.). Polysorbate 80NF, HLB=15.0 (Tween 80) was purchased from Advance Scientific & Chem.(Ft. Lauderdale, Fla.). Isopropyl myristate (IPM), oleyl alcohol (99%),anhydrous ethyl alcohol, sorbitan mono-oleate (Span 20), HLB=8.6, andphosphate buffered saline tablets (PBS) were purchased from Sigma (St.Louis, Mo.). HPLC grade solvents were used as received. Skin: Humancadaver skin from the chest, back, and abdominal regions was obtainedfrom the National Disease Research Institute (Philadelphia, Pa.). Theskin was stored at −80° C. until use.

C. Methods

(i) Microemulsion Phase Diagrams. Four microemulsion (ME) systems wereinvestigated to determine their ternary phase diagrams. All percentagesare given as mass ratios. Organic System Aqueous Phase Phase SurfactantPhase 1 H₂O:Ethanol (1:1) IPM Tween 80 2 H₂O:Ethanol (1:1) IPM Tween80:Span 20 (49:51) 3 H₂O IPM Tween 80:Ethanol (1:1) 4 H₂O IPM Tween80:Ethanol (2:1)Each of the three components for a system was titrated until a phasechange between microemulsion and two phase mixture was observed. Theboundary of this transition was recorded over the entire concentrationrange. A microemulsion was determined as a miscible, optically clear,stable solution. At the transition to a two phase regime, there is aclear clouding of the mixture as well as an eventual separation of thephases. All microemulsion systems were stable for over 6 months.

(ii) Preparation of Formulations. Sample solutions were prepared in 20ml glass vials and saturated with drug. Drug flux was tested through MEsystem 1 at two selected concentrations, one in the W/O region, and theother in the O/W. The W/O system consisted of H₂O:IPM:Tween 80 (10:52:38w/w) while the O/W system contained H₂O:Ethanol:IPM:Tween 80(27:18:16:39 w/w). Both systems stably incorporated 10% w/w NMP and 10%w/w oleyl alcohol. Drug concentration in the formulation was generally4% for lidocaine, 2% for diltiazem HCl, and 0.4% for estradiol. The“water phase” sample consisted of the aqueous elements H₂O:Ethanol:NMP(51:31:18) in the same relative proportions as if the organic componentswere removed. All vehicles studied formed miscible, single phaseliquids.

(iii) Lidocaine Partitioning. The logarithm of the relative partitioncoefficient between IPM and water (log[IPM/H₂O]) was determined for NMPconcentrations of 0-35% (v/v). In a micro-centrifuge tube, 500 μl of IPMwas added to 500 μl of ddH₂O with the addition of the appropriate amountof NMP. Lidocaine free base was included at 1.0 mg/ml in the organic(IPM) phase. For lidocaine HCl samples, the drug was dissolved in theaqueous phase at 1.0 mg/ml. The two phase system was thoroughly vortexedand allowed to equilibrate. The samples were then centrifuged at 14,000rpm for 6 minutes to separate the phases. The concentration of lidocainein each phase was determined by HPLC.

(iv) Preparation of Skin Samples. Human cadaver skin was thawed at roomtemperature. The epidermis-SC was separated from the full thicknesstissue after immersion in 60° C. water for 2 minutes. Heat stripped skinwas immediately mounted on diffusion cells.

(v) Skin Transport Experiments. The skin was mounted onto a side-by-sideglass diffusion cell with an inner diameter of 5 mm. The two halves ofthe cell were clamped shut and both reservoirs were filled with 2 ml ofphosphate buffered saline (PBS, 0.01 M phosphate, 0.137 M NaCl, pH 7.4).The integrity of the skin was verified by measuring the electricalconductance across the skin barrier at 1 kHz and 10 Hz at 143.0 mV (HP33120A Waveform Generator). Skin samples measuring 4-14 μA at 1 kHz wereused for the diffusion studies. Prior to introducing the donor solution,the skin sample was thoroughly rinsed with PBS to remove surfacecontaminants. At t=0, the receiver compartment was filled with 2.0 ml ofPBS, while 2.0 ml of sample was added to the donor compartment. Bothcompartments were continuously stirred to maintain even concentrations.At regular time intervals, 1.0 ml of the receiver compartment wastransferred to a glass HPLC vial. The remaining solution in the receivercompartment was thoroughly aspirated and discarded. Fresh PBS (2.0 ml)was dispensed into the receiver compartment to maintain sink conditions.At 21 hours, the experiment was terminated. After both compartments wererefilled with PBS, the conductance across the skin membrane was againchecked to ensure that the skin was not damaged during the experiment.All flux experiments were conducted in triplicate at room temperature.The observed variability of the individual drug transport values wasconsistent with the previously established 40% intersubject variabilityof human skin.⁹⁰⁹⁰Williams, A. C.; Cornwell, P. A.; Barry, B. W. Int. J. Pharm. 1992,86, 69-77

(vi) Drug Quantification. Lidocaine was assayed by high pressure liquidchromatography (Shimadzu model HPLC, SCL-10A Controller, LC-10AD pumps,SPD-M10A Diode Array Detector, SIL-10AP Injector, Class VP v.5.032Integration Software) on a reverse phase column (Waters μBondapak™ C₁₈3.9×150 mm) using ddH₂O (5% acetic acid, pH 4.2)/acetonitrile (35:65v/v) as the mobile phase, under isocratic conditions (1.6 mL/min) bydetection at 237 nm. The retention time of lidocaine under theseconditions was between 3.4 and 4.3 minutes. Standard solutions were usedto generate calibration curves. Diltiazem HCl was quantified on a WatersSymmetry® C₁₈ 5 μm, 3.9×150 mm column (WAT046980). The mobile phaseconsisted of aqueous phase:acetonitrile:methanol (50:25:25) where theaqueous phase consisted of 1.16 g/L d-10-camphorsulfonic acid, 0.1 Msodium acetate, pH 6.2. The system ran at a flow rate of 1.6 ml/min.Chromatograms were integrated at a peak of 240 nm. Estradiol wasquantified on a Waters 4.6×250 mm C₁₈ column. The mobile phase consistedof acetonitrile:water (55:45) at a flow rate of 2.0 m/min. Chromatogramswere integrated at a peak of 280 nm.

(vii) Calculations. The total mass of drug transported across the skinwas determined by HPLC. The flux equation gives:$J = {{\frac{1}{A}( \frac{\mathbb{d}M}{\mathbb{d}t} )} = {P\quad\Delta\quad C}}$where J is flux (μg cm⁻² hr⁻¹), A is cross sectional area of the skinmembrane (cm²), P is the apparent permeability coefficient (cm hr⁻¹),and ΔC is the concentration gradient. In this experiment, ΔC is taken asthe saturation concentration (given infinite dose and sink conditions),and dM/dt is averaged as the total mass transport over the steady stateportion of the transport curve. Statistical analyses were performed bythe Student's t-test.D. Results and Discussion

(i) Microemulsion systems. Thermodynamically stable, opticallytransparent, single phase, liquid formulations were created with thefour systems (FIGS. 20-23). An ethanol co-surfactant is necessary tomaintain stable O/W emulsions. This is consistent with previous workwith ME systems where co-surfactants (usually short chain alcohols) arenecessary to maintain a single phase.⁹¹ In system 2, a combination oftwo nonionic surfactants was used. The mixture of 49:51 w/w Tween 80(HLB=15) and Span 20 (HLB=8.6) has been reported to act in synergy tomaximize water uptake.⁹² Although this system was not tested fortransdermal transport, the phase diagram does indeed indicate that MEformation occurs at lower surfactant concentrations. The phase diagramsin FIGS. 22-23 contain the same components as FIG. 20. In these twodiagrams, the surfactant/cosurfactant (Tween 80/ethanol) ratio is fixedover the entire range. It is apparent that having too much ethanol isdetrimental to ME formation (Table 11). The maximum IPM uptake in O/W MEsystems occurs at Tween 80/ethanol ratio of 1:1. Furthermore, it wasobserved that the cosurfactant was necessary primarily to stabilize MEformulations with high water content. Systems with too little ethanolwere unable to form stable O/W microemulsions.⁹¹Ho, Hsiu-O; Hsiao, Chih-Chuan; Sheu, Ming-Thau. J. Pharm. Sci. 1996,85(2) 138-143⁹²Huibers, Paul D. T. Surfactant Self-Assembly, Kinetics andThermodynamics of Micellar and Microemulsion Systems. Ph.D. Thesis,University of Florida, 1996

All systems could stably incorporate 10% w/w of the transdermalenhancers NMP, oleyl alcohol, oleic acid, or decanoic acid. Drugsolubility reached ˜30% w/w lidocaine free base in the W/O system and˜25% lidocaine HCl in the O/W system. With such high tolerance for theaddition of both hydrophilic and hydrophobic molecules, the ME systemsstudied are robust vehicles for transdermal drug delivery.

(ii) Transdermal Transport. A W/O and O/W formulation from system 1 wasselected to test transdermal delivery of hydrophilic and hydrophobicdrugs across stripped human skin. The results (Table 12) indicate thatthe O/W system provided significantly better flux for all the drugsstudied (p<0.025). The presence of a second drug in the same ME(estradiol with diltiazem HCl) did not affect the transport of eitherdrug (p>0.5). The permeability of drug from the water phase solution isstatistically comparable to that of the O/W ME formulation (p>0.25).

For all the drugs tested, the ME systems provided significantenhancement (Table 14). The finding that flux is improved in O/Wformulations as compared with W/O systems even for the hydrophobic drugssuggests that transport from the aqueous phase is key. When the organicphase and surfactants were removed from the ME, leaving only the waterphase components (H₂O, ethanol, NMP), the flux was comparable to thatfrom the O/W ME (Table 13). Previous work indicates that the H₂O/NMPsynergy provides greater transdermal flux enhancement thanH₂O/ethanol.¹⁷ Although the complexity of the multiple components in thesystem makes it difficult to determine the exact molecular interactions,it appears that the presence of NMP in the water phase plays a key rolein the transport of hydrophobic drugs from an O/W ME.¹⁷Ranade, Vasant V. J. Clin. Pharmacol. 1991, 31, 401-418

It has been previously suggested that ME transdermal enhancement is aresult of increasing drug concentration in the donor phase. In oursystems containing the chemical enhancer NMP, we believe that theeffective permeability of the membrane is also affected. If enhancementis merely a concentration effect, then the permeability of drug acrosshuman skin should remain constant. The permeability of all four drugswas compared from the ME systems against the solvent (IPM or H₂O) inwhich they were most soluble (Table 14). There is a clear permeabilityenhancement for both hydrophilic and hydrophobic drugs from the MEsystems (p<0.001). This finding agrees with previous work where we foundthat NMP is capable of improving permeability of drugs from both IPM andH₂O.^(16,17)¹⁶Sleight, P. J. Cardiovasc. Pharmacol. 1990, 16, Suppl 5: S113-119¹⁷Ranade, Vasant V. J. Clin. Pharmacol. 1991, 31, 401-418

(iii) Effect of NMP on Lidocaine Partitioning. NMP is freely miscible inboth H₂O and IPM. It is also capable of improving lidocaine partitioninginto the phase where the drug is less soluble (FIG. 26). The hydrophobiclidocaine free base partitions 2.6 times more in the aqueous phase withthe addition of 33% NMP. Similarly, the hydrophilic lidocaine HClpartitions 6.5 times more favorably in the IPM phase with the additionof 33% NMP. The concentrations of drug in the minority phase is improved1.9-fold for lidocaine free base and 5.7-fold for lidocaine HCl. Fromthese results, we can conclude that NMP can act as a partition enhancerin ME systems. In our model for hydrophobic drug transport from an O/WME, the drug (e.g. lidocaine free base) must first partition from theorganic phase into the aqueous phase to reach the skin. The presence ofNMP in the system is able to increase the concentration of thehydrophobic drug in the water phase, making it available for transport.Data from FIG. 26 indicates that NMP is also capable of improving thepartitioning of hydrophilic drugs to the IPM phase in a W/O ME.

(iv) O/W ME Systems. We propose the following mode of enhancement by NMPin the O/W system. A hydrophobic drug will preferentially partition inthe encapsulated organic phase, making flux difficult. The presence ofNMP improves partition (and concentration) in the bulk aqueous phase.While in this phase, the drug can favorably partition into the skin withthe aid of NMP.¹⁷ For hydrophilic drugs, the presence of NMP in theaqueous phase improves the permeability of the drug across human skinvia H₂O/NMP synergy.¹⁷ The role of the organic phase for hydrophilicdrug transport from an O/W ME is unknown.¹⁷Ranade, Vasant V. J. Clin. Pharmacol. 1991, 31, 401-418

We further hypothesize that NMP is a more effective enhancer from theaqueous phase of a ME than the organic phase. NMP was found to have anIPM/H₂O partition ratio of 0.02. Because NMP resides almost exclusivelyin the water phase of the system, its enhancing effects from that phaseshould dominate. In a W/O ME, the NMP is sequestered in the encapsulatedphase and unable to interact with the skin. This might explain why boththe hydrophilic and hydrophobic drugs transport better from the O/W ME.A second mode of hydrophobic drug flux enhancement by NMP from the waterphase is also possible. Hydrophobic molecules will not readily leave anorganic phase in which they are highly soluble. For this reason, thepartition of lidocaine free base from IPM into the skin is slow.However, when lidocaine is in the aqueous phase, it has two partitioningoptions. It can return to the organic phase, or follow NMP (to which ithas high affinity) across the skin membrane. By this account, the waterphase of an O/W ME provides a favorable environment for a hydrophobicdrug to partition into the skin.

The transdermal delivery of diltiazem HCl has not previously beenreported. A drug such as diltiazem HCl is normally precluded fromtransdermal delivery. Its large molecular weight greatly diminishes itspermeability across the skin.⁹³ Ionic drugs have also been proven to bedifficult to deliver transdermally.^(94,95,96) Transport of diltiazemHCl from the O/W ME system showed the most drastic enhancement of the 4drugs tested. This result is promising for delivery of other ionic saltdrugs from the ME system.⁹³Mitragotri, Samir; Johnson, Mark E.; Blankschtein, Daniel; Langer,Robert. Biophysical Journal. 1999, 77, 1268-1283.⁹⁴Gorukanti, Sudhir R.; Li, Lianli; Kim, Kwon H. Int. J. Pharm. 1999,192, 159-172⁹⁵Roy, Samir D.; Roos, Eric; Sharma, Kuldeepak. J. Pharm. Sci. 1994, 83,126-130⁹⁶Peck, Kendall D.; Ghanem, Abdel-Halim; Higuchi, William I. J. Pharm.Sci. 1995, 84, 975982

The systems studied provide many interesting characteristics for atransdermal delivery vehicle. They are robust, and stable to theaddition of significant amounts of soluble enhancers or excipients. Theyare capable of enhancing both hydrophilic and hydrophobic drugs, as wellas simultaneous delivery of two drugs without diminished flux. The MEsystems are also thermodynamically stable, and transport of lidocainefree base after 6 months storage at room temperature was equivalent toits initial value. We believe the novel systems proposed in this studyoffer a viable vehicle for transdermal drug delivery.

E. Tables TABLE 11 Maximum IPM Uptake in O/W ME Systems 2. Tween80/Ethanol % IPM % Tween Ratio Uptake (w/w) 80/Ethanol (w/w) 1:2 8.3 662:3 8.1 65 1:1 47 50 2:1 42 53 4:1 4.5 56 9:1 1.6 58

TABLE 12 Lidocaine Free Base and Lidocaine HCl Transport from ME SystemsLidocaine Free Base Lidocaine HCl Flux_(ss) Permeability Flux_(ss)Permeability 3. Formulation (μg/cm²/hr) (cm/hr · 10⁵) (μg/cm²/hr) (cm/hr· 10⁵) Water  6.0 ± 1.0 133 ± 23 0.61 ± 0.38 0.61 ± 0.38 W/O ME 16.5 ±1.8 40.2 ± 4.5 2.1 ± 0.2 3.5 ± 0.3 O/W ME 23.3 ± 1.3 75.8 ± 4.1 10.2 ±3.9  18.1 ± 6.9 N = 3

TABLE 13 Estradiol and Diltiazem HCl Transport from ME Systems EstradiolDiltiazem HCl For- Flux_(ss) Permeability Flux_(ss) Permeabilitymulation (μg/cm²/hr) (cm/hr · 10⁵) (μg/cm²/hr) (cm/hr · 10⁵) H₂O 0.015 ±0.006 460 ± 183 0.05 ± 0.01 0.015 ± 0.004 W/O ME 0.053 ± 0.029 1.1 ± 0.60.25 ± 0.13 1.2 ± 0.6 W/O ME 0.12 ± 0.06 2.4 ± 1.2 0.24 ± 0.08 1.2 ± 0.4Both Drugs O/W ME 0.27 ± 0.07 5.8 ± 1.5 1.6 ± 0.3 7.8 ± 1.3 O/W ME 0.23± 0.05 5.0 ± 1.2 1.6 ± 0.4 7.8 ± 1.9 Both Drugs Water 6.5 ± 1.7 6.1 ±3.7 PhaseN = 3

TABLE 14 Permeability Enhancement of ME Systems Permeability (cm/hr ·10⁵) Enhancement Estradiol IPM <0.1 W/O ME 1.1 ± 0.6 >11 O/W ME 5.8 ±1.5 >58 Diltiazem HCl H₂O 0.015 ± 0.004 W/O ME 1.2 ± 0.6 80 O/W ME 7.8 ±1.3 520 Lidocaine Free Base IPM 7.2 ± 1.1 W/O ME 40.1 ± 4.5  5.6 O/W ME123 ± 36  17 Lidocaine HCl H₂O 0.61 ± 0.38 W/O ME 3.5 ± 0.3 5.7 O/W ME18.1 ± 6.9  30N = 3F. References for Example 4

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1. A transdermal delivery system comprising a drug formulated with atransport chaperone moiety that reversibly associates with the drug,said chaperone moiety being associated with the drug in the formulationso as to enhance transport of the drug across dermal tissue andreleasing the drug after crossing said dermal tissue.
 2. The system ofclaim 1, wherein the transport chaperone is n-methyl pyrrolidone (NMP),octadecene, isopropyl myristate (IPM), oleyl alcohol, oleic acid or aderivative thereof.
 3. The system of claim 1, wherein the drug is alidocaine, a prilocaine, an estradiol or a diltiazem.
 4. The system ofclaim 3, wherein the drug is a free base.
 5. The system of claim 1,wherein the drug is lidocaine HCl, lidocaine free base, prilocaine HCl,estradiol or diltiazem HCl.
 6. The system of claim 1, wherein thechaperone moiety and drug are associated by ionic, hydrophobic,hydrogen-bonding and/or electrostatic interactions.
 7. A microemulsionsystem for transdermal delivery of a drug, which system solubilizes bothhydrophilic and hydrophobic components.
 8. The microemulsion system ofclaim 7, being a cosolvent system including a lipophilic solvent and anorganic solvent.
 9. The microemulsion system of claim 8, wherein thecosolvents are NMP and IPM.
 10. The microemulsion system of claim 7,having an aqueous phase of water and ethanol, an organic phase ofisopropyl mystate (IPM) and a surfactant phase of Tween
 80. 11. Themicroemulsion system of claim 10, wherein the aqueous phase is water andethanol, the organic phase is IPM, and the surfactant phase is Tween 80and Span
 20. 12. The microemulsion system of claim 7, having an aqueousphase, a hydrophobic organic phase, and a surfactant phase.
 13. Themicroemulsion system of claim 7, wherein the system is a water-in-oilsystem.
 14. The microemulsion system of claim 7, wherein the system isan oil-in-water system.